Abstract—Photoaffinity labeling of synthetic DN As with ethidium monoazide was studied to determine if the efficiency of adduct formation was related to DNA sequence. Equilibrium drug binding to DNA homopolymers and copolymers was quanitified by phase partition techniques. The amount of drug bound to a deoxypolymer at equilibrium was then compared to the fraction of ethidium analog covalently‐linked following photoactivation at the same drug/DNA input ratio. There were significant sequence‐related differences in the ability of the photoaffinity probe to label DNA covalently. The efficiency of covalent‐adduct formation decreased in the order poly(dG‐dC).poly(dG‐dC)>poly‐(dG). poly(dC)poly(dA‐dT). poly(dA‐dT)poly(dA). poly(dT). Ethidium monoazide was about 2‐fold more efficient in labeling deoxyhomopolymers and deoxycopolymers composed of G‐C pairs than the A‐T base counterparts. In low ionic buffers (0.015MNa+), the efficiency of photoactivation decreased with increasing ethidium monoazide concentrations. However. the base sequence effect was observed over a 40‐fold range of drug concentrations. Therefore, the amount of ethidium monoazide bound to a DNA site after irradiation does not appear to represent the true affinity