Tetradecanoyl phorbol acetate (TPA) has been shown to inhibit 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced mouse P450IA1 benzo[a]pyrene hydroxylase activity (Raunio, H., and Pelkonen, O. (1983) Cancer Res. 43, 782-786). When we co-administered TPA and TCDD to C57BL/6 mice, the accumulation of TCDD-inducible liver P450IA1 and P450IA2 mRNA, as well as kidney P450IA1 mRNA, was greatly inhibited. When nuclear run-on assays were conducted, maximal levels of transcriptional activation were achieved for both liver Cyp1a-1 and Cyp1a-2 with 1 micrograms/kg (approximately equal to 3.0 nmol/kg) TCDD. TCDD elicited a dose-dependent increase in the rates of gene transcription, which paralleled the induction of P450IA1 and P450IA2 mRNA. Only Cyp1a-1 gene transcription was elevated in kidney. When these experiments were repeated following the co-administration of TPA with TCDD, the levels of TCDD-mediated transcriptional increases in liver Cyp1a-1 and Cyp1a-2 and P450IA1 and P450IA2 mRNAs were dramatically inhibited. The reduction in Cyp1a gene transcription by TPA could be accounted for by reduced DNA binding of the dioxin receptor to the xenobiotic-responsive element (XRE) sequences, as measured by gel-retardation analysis. Analysis of nuclear [3H]TCDD dioxin receptor by sucrose density gradients demonstrated that the inhibition of Cyp1a gene transcription and DNA binding by TPA resulted from a reduction in nuclear dioxin receptor concentration.