Mouse liver cytosol contains saturable, high-affinity binding sites for the aromatic carcinogen benzo[a]pyrene that are distinct from the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-binding aryl hydrocarbon receptor. Specific binding parameters determined by an equilibrium binding assay indicate a dissociation constant of 7.7 nM and a binding capacity of 4.7 pmol of benzo[a]pyrene per milligram of cytosolic protein. Although the data best fit a single class of binding sites by Scatchard and Hill analyses, discrete 4 S and 9 S [3H]benzo[a]pyrene peaks are identified on sucrose density gradients comprising about 98% and 2% of the specific binding, respectively. Steroid hormones and other ligands for previously described binding proteins have no effect on the specific carcinogen binding when present in the cytosol incubation at saturating levels. The glutathione S-transferases, shown in rat and human liver to possess carcinogen-binding properties, were also found not to be responsible for this receptor-like benzo[a]pyrene binding in mouse liver cytosol. Competition binding studies indicate that other aromatic hydrocarbon compounds of a structure similar to that of benzo[a]pyrene are equipotent in affinity for this major carcinogen-binding site.