We reported previously that protein associated with Myc (PAM) interacts with the C2 domain of type V adenylyl cyclase (ACV-C2) and that purified PAM is a potent inhibitor of Galphas-stimulated ACV activity (J Biol Chem 276:47583-47589, 2001). The present study was conducted to identify the region in PAM that inhibits ACV activity and to determine whether its binding with the ACV-C2 is necessary and sufficient to inhibit the enzyme. Coexpression of ACV and full-length PAM or its N-terminal third (PAM-N) in COS-7 cells inhibited isoproterenol-stimulated cAMP accumulation. Deletion of the RCC1 homology domains in PAM-N abolished its ability to inhibit isoproterenol-stimulated cAMP formation in cells. Purified GST fusion protein of the second RCC1 homology domain (RHD2) of PAM was sufficient to bind with ACV-C2 and inhibit Galphas-stimulated ACV activity. In addition, deletion of 11 amino acids in GST-RHD2 obliterated its ability to bind with and inhibit ACV. The C terminus of the RHD2 domain bound with ACV-C2 without inhibiting enzyme activity. Furthermore, substitution of His912 and His913 with alanine in the GST-RHD2 obliterated its ability to inhibit ACV without altering binding to ACV-C2. Likewise, H912/913A mutants of both PAM-N and full-length PAM did not inhibit cAMP formation in cells. Thus, the RHD2 domain of PAM is sufficient to inhibit Galphas-stimulated ACV activity and the binding of RHD2 to ACV-C2 is necessary but not sufficient for this inhibition. Moreover, His912 and His913 in PAM are critical for inhibiting ACV.