Previous spectroscopic studies of the major adduct formed by reaction of (+/-)-7r,8t-dihydroxy-9t, 10t-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE-I) with linear DNA have been interpreted to suggest that the adduct is not intercalated in the double helix. However, studies of the electrophoretic mobility of supercoiled DNA treated with BPDE-I suggest that the adduct is intercalated. To resolve these interpretations, we have studied the reaction of BPDE-I with supercoiled and linear DNA. The kinetics of DNA-catalyzed hydrolysis and of covalent binding are similar for the two DNAs; supercoiled DNA exhibits a 20% increase in the rate of hydrolysis of BPDE-I at low DNA concentration compared to linear DNA. Fluorescence excitation spectra and fluorescence quenching experiments provide no support for a model in which BPDE-I adducts are intercalated in supercoiled DNA. When deoxyribonucleoside adducts were analyzed by high-performance liquid chromatography, identical distributions of BPDE-I adducts were found for supercoiled and linear DNA. These data are consistent with a previously proposed model (Hogan, M. E., Dattagupta, N., and Whitlock, J.P., Jr. J. Biol. Chem., 256: 4504-4513, 1981; Taylor, E.R., Miller, K. J., and Bleyer, A. J. J. Biomol. Struct. Dyn., 1: 883-904, 1983), in which the major BPDE-I adduct in both linear and supercoiled DNA exists in a conformation which allows stacking with the neighboring base pair and introduces a "kink" into the path of the helical axis. Although this model provides an explanation for all available experimental data, there are undoubtedly other DNA adduct conformational models which are also consistent with the data.