Cytochrome P450 monooxygenases or CYPs, a family of endobiotics and xenobiotics metabolizing enzymes, are found in all organisms. We reported earlier that the promoters of Drosophila Cyp6a2 and Cyp6a8 genes are induced by caffeine. Since caffeine antagonizes adenosine receptor (AdoR) and inhibits cAMP phosphodiesterase (PDE), we used luciferase reporter gene to examine whether in SL-2 cells and adult Drosophila, induction of the two Cyp6 genes is mediated via AdoR and/or PDE pathway. Results showed that AdoR is not involved because AdoR agonists or antagonists do not affect the Cyp6 promoter activities. However, inhibition of PDE by specific inhibitors including caffeine causes induction of both Cyp6 gene promoters. We also found that flies mutant for dunce gene coding for cAMP-PDE, have higher Cyp6a8 promoter activity than the wild-type flies. We demonstrate that caffeine treatment increases intracellular cAMP levels, and cAMP treatment induces the Cyp6 gene promoters. Since both Cyp6 genes have multiple sites for JUN transcription factors, which generally play a positive role in cAMP pathway, effect of Drosophila jun (D-jun) on the Cyp6a8 promoter activity was examined. Results showed that the expression of D-jun sense plasmid causes downregulation rather than activation of the Cyp6a8 promoter. Conversely, expression of antisense plasmid increased the promoter activity. Interestingly, caffeine treatment decreased the D-JUN protein level in SL-2 cells as well as in adult flies. These results suggest that D-jun acts as a negative regulator, and caffeine induction of Cyp6a8 and Cyp6a2 genes is mediated by the upregulation of cAMP pathway and downregulation of the D-JUN protein level.