CYP1B1 is unique among P450 cytochromes in exhibiting inductive responses mediated by both the Ah receptor (AhR) and cAMP. cAMP induction was mediated either by a 189bp far upstream enhancer region (FUER, -5110 to -5298) or by a 230bp AhR-responsive enhancer region (AhER) (-797 to -1026). CYP1B1 luciferase reporters respond selectively to cAMP and TCDD in adrenal Y-1 cells (only cAMP), testis MA10 cells (cAMP>TCDD), and C3H10T1/2 mouse embryo fibroblasts (only TCDD). In Y-1 cells, which lack AhR, cAMP induction is totally dependent on the FUER, including absolute requirements for upstream and downstream halves of this region, and for CREB activity at a CRE sequence located at the 3(')-end. cAMP stimulation of the FUER was remarkably high (27-fold) and equally effective when linked to an HSV-TK promoter, indicating direct cAMP activation of the FUER. Binding of CREB to the essential CRE was demonstrated along with dominant negative effects of functionally impaired mutants. cAMP induction in MA10 cells was partially mediated by the FUER mechanism but was regulated additionally by AhER through AhR activity. MA10 cells also exhibit cAMP-dependent AhR down-regulation and AhR/Arnt complex formation. Mutations in AhER including XRE5 were similarly inhibitory to cAMP stimulation in MA10 cells and to TCDD stimulation in C3H10T1/2 cells. Transfection of AhR into the AhR-deficient Y-1 cells did not introduce this second mechanism, which indicated a need for additional components that are present in MA10 cells.