In the heart, the rapid propagation and synchronization of action potentials necessary for a normal heart rhythm and an effective cardiac output are mediated by specialized ionic channels that link adjacent cells and are known collectively as gap junctions. Cardiac gap junctions are gated by various physiological and pharmacological agents, but the role of voltage in their gating is unclear. Whereas embryonic or neonatal ventricular cells have voltage-gated gap junctions, adult cells are reported to have only voltage-independent gap junctions. We studied the voltage dependence of adult rat atrial gap junctions by individually voltage clamping each cell of a connected cell pair and controlling the transjunctional voltage (Vj), measuring transjunctional current (Ij), and calculating junctional conductance (gj). Two distinct populations of cell pairs were observed: highly coupled pairs with the peak gjs ranging from 3.4 to 40 nS and weakly coupled pairs with the peak gjs ranging from 0.3 to 2.0 nS. gjwas dependent on Vj, and Ijdecayed exponentially, with the time constants being voltage dependent. Voltage dependence was most apparent when cells were poorly coupled. The gjdid not decrease to zero. The normalized conductance-Vjplot was fit with a two-state Boltzmann model as a first approximation, resulting in a half-inactivation potential and gating charge of 42.5 mV and 1.14 eV, respectively, for the weakly coupled cell pairs. For highly coupled cell pairs, the half-inactivation potential shifted to 53.3 mV. Single gap junctional channels had a gj of 36.2±7.6 pS (range, 27–49 pS), which was Vjindependent. The junctional current as obtained by ensemble average of single-channel recordings has a time constant of current decay comparable to that observed for Ijat similar Vj. These findings suggest that the voltage-dependent kinetics of Ijresult from the all-or-none gating of a population of 36-pS conductance channels. It is interesting to speculate that voltage gating of gap junction channels provides rapid fine control of cell-to-cell interaction, particularly in poorly coupled cells, and importantly determines both cardiac excitability and impulse propagation.