Peptides derived from various regions of type V adenylyl cyclase (AC) were studied to determine their effect on AC catalytic activity. Out of 10 examined, only one peptide, peptide 2 (Lys425-Cys444), significantly inhibited both basal and stimulated (forskolin or GTP gamma S (guanosine 5'-O-thiotriphosphate)) type V AC catalytic activity overexpressed in CMT cells. The sequence of this peptide was taken from the first cytoplasmic domain of type V AC, which has a high sequence homology to other AC isoforms. Competition studies performed between the peptide and the substrate ATP showed that the inhibition was noncompetitive. Mutation or truncation of the peptide designed to destroy its secondary structure totally negated the inhibitory effect. This peptide also inhibited the catalytic activity of purified types II and V AC, as well as that of various cells, including S49 cyc- cells. Our data indicate that the peptide directly interacts with AC to inhibit catalytic activity; this provides new information regarding regions of the enzyme involved in its catalytic activation.