Using the yeast two-hybrid assay and the second of the two large cytosolic domains of type V adenylyl cyclase (ACV) as bait, we identified a small region (amino acids 1028-1231) in the protein associated with Myc (PAM) as an interaction site for ACV. This small region of PAM as well as purified full-length PAM inhibited the activity of ACV. Additionally, full-length PAM was a very potent inhibitor of ACI and AC activities in S49 cyc(-) cells and HeLa cells with IC(50) values in the pm and low nm range. Moreover, the regulator of chromatin condensation 1-like domain of PAM (amino acids 446-1062) was sufficient and as potent as full-length PAM at inhibiting the activity of ACV. Interestingly, full-length PAM did not inhibit ACII activity that was stimulated by either forskolin of Galpha(s). When endogenous levels of PAM in HeLa cells were decreased using antisense oligodeoxynucleotides, the basal cAMP content was elevated, and the dose-response curve for vasoactive intestinal peptide-elicited cAMP accumulation in HeLa cells was shifted to the left. Therefore, we conclude that PAM is a very potent, novel inhibitor of specific isoforms of AC. Furthermore, the regulator of chromatin condensation 1-like domain of PAM is sufficient to exert the effects of the full-length protein on AC and decreases in endogenous PAM levels in HeLa cells can modulate both basal and agonist stimulated cAMP accumulation.