When benzo[a]pyrene (B[a]P) was administered intraperitoneally to rats 48 hr before they were killed, the DNA-synthesizing capability of isolated rat liver nuclei was decreased as compared with control animals. B[a]P also inhibited in vitro DNA synthesis in nuclei purified from control animals; this effect was enhanced by NADPH. DNA polymerases solubilized from purified nuclei of B[a]P-treated animals were less active than those of control animals. DNA polymerase alpha was more inhibited than DNA polymerase beta. Purified rat liver nuclei devoid of cytoplasmic contamination possess an NADPH-dependent B[a]P hydroxylase activity. The observed inhibition of DNA synthesis in nuclei isolated from B[a]P-treated rats was increased by NADPH. Moreover, there was an increased inhibition of DNA polymerase activity by nuclear membranes obtained from B[a]P-treated animals when the incubations were performed in the presence of NADPH. Also, the derivative B[a]P-trans-9,10-dihydrodiol was a potent inhibitor of DNA polymerase alpha under conditions where DNA polymerase beta was less affected. These results suggest that nuclear B[a]P hydroxylase might be involved in the inhibition of DNA synthesis probably at the level of DNA polymerase alpha. As in the in vivo studies, the nuclear polymerase most affected by the hydrocarbon in vitro was DNA polymerase alpha.