Carcinogenic doses of 7,12-dimethylbenz(α)anthracene (DMBA) failed to induce mammary carcinoma in the rats that have received N6,O2′-dibutyryl cyclic adenosine 3′,5′-monophosphate (DBcAMP) (Cancer Res., 43: 2736, 1983). We now report that the anticarcinogenic effect of DBcAMP correlates with its effect on DNA binding of the carcinogen and on gene expression. Cultured mammary epithelial cells in exponential phase of growth were used to determine the effect of DBcAMP on DMBA binding to DNA. DBcAMP inhibited the DMBA binding in carcinogen-susceptible mammary cells of 50-day-old rats, but it had no effect on the binding in the DMBA-resistant mammary cells of 35- and 110-day-old rats. The inhibitory effect of DBcAMP was appreciable at the concentration of 10-7 m, one tenth the concentration of [3H]DMBA. DBcAMP at 10-6 m concentration exhibited the maximal inhibition of DMBA binding; i.e., binding in DMBA-susceptible mammary cells was reduced to the level of binding observed in DMBA-resistant mammary cells. Polyadenylate-containing RNAs isolated from mammary glands of DMBA-susceptible rats (50-day-old) yielded translation products in vitro which bear a greater resemblance to translation products of growing DMBA-induced tumors than they do to products of messenger RNAs from regressing tumors or DMBA-resistant mammary glands of 110-day-old rats. DBcAMP administered to the susceptible rats resulted in changes in the translation products of the mammary glands; the translation products of the glands became similar to those of DMBA-resistant mammary glands or regressing tumors. DMBA feeding 1 day after DBcAMP treatment could not reverse this effect of DBcAMP. These data suggest that the role of cyclic adenosine 3′,5′-monophosphate at the genomic level is responsible for the anticarcinogenesis of mammary cells.