AbstractTransient electric birefringence has been used as an analytical tool to study the orientation of DNA in agarose gels, and to study the orientation of the matrix alone. The sign of the birefringence of DNA oriented in an agarose gel is negative, as observed in free solution, indicating that the DNA molecules orient parallel to the direction of the electric field. If the median pore diameter of the gel is larger than the contour length of the DNA molecule, the DNA effectively does not see the matrix and the birefringence relaxation time is the same as observed in free solution. However, if the median pore diameter of the gel is smaller than the contour length of the DNA, the DNA molecule becomes stretched as well as oriented. For DNA molecules of moderate size (⩽ 4 kb), stretching in the gel causes the birefringence relaxation times to increase to the values expected for fully stretched molecules. Complete stretching is not observed for larger DNA molecules. The orientation and stretching of DNA molecules in the gel matrix indicates that end‐on migration, or reptation, is a likely mechanism for DNA electrophoresis in agarose gels.When the electric field is rapidly reversed in polarity, very little change in the orientation of the DNA is observed if the DNA molecules were completely stretched and had reached their equilibrium orientation before the field was reversed in direction. Hence completely stretched, oriented DNA molecules are able to reverse their direction of migration in the electric field with little or no loss of orientation. However, if the DNA molecules were not completely stretched or if the equilibrium orientation had not been reached, substantial disorientation of the DNA molecules is observed at field reversal. The forced rate of disorientation in the reversing field is faster than the field‐free rate of disorientation. Complicated patterns of reorientation can be observed after field reversal, depending on the degree of orientation in the original field direction.The effect of pulsed electric fields on the orientation of the agarose gel matrix itself was also investigated. If very short pulses of high amplitude (e.g.1–10 kV/cm in amplitude and 10–1000 μs in duration) were applied to the gel, the sign of the birefringence was small and positive, the Kerr law was obeyed, and the birefringence decayed to zero after the removal of the electric field with a relaxation time varying from 10–220 μs, depending on the length of the orienting pulse. These results indicate that individual agarose chains or bundles of chains, or dangling ends of the matrix, could be oriented by very short pulses of high amplitude. However, when smaller electric fields were used (e.g., 10–100 V/cm applied to the gel for 0.5–2 s), the amplitude of the birefringence of the gel matrix increased markedly and the signal passed through an extremum several seconds after removal of the electric field before decaying slowly to zero. These slow time‐dependent effects indicate that domains in the agarose matrix were being oriented by the longer pulses used at the lower electric field strengths. The sign of the birefringence of the agarose gel could be reversed from positive to negative, orvice versa, by reversing the direction of the applied electric field, indicating that the domains apparently change their direction of orientation from parallel to perpendicular (orvice versa) after field reversal. Orientation and reorientation of microdomains of the matrix in alternating pulsed electric fields would increase the fluidity of the matrix, making it easier for very large DNA molecules to migrate through the gel dur