C-type natriuretic peptide (CNP) and brain natriuretic peptide (BNP) are members of the natriuretic peptide family, which have been shown to interact with ANP-C/ANF-R2 receptors in addition to ANP-B receptor subtypes. The present study was undertaken to investigate if the interaction of CNP and BNP with ANP-C receptors results in the inhibition of adenylyl cyclase activity. CNP and BNP inhibited adenylyl cyclase activity in heart and brain striatal membranes in a concentration dependent manner with an apparent Ki between 0.1 and 1.0 nM. Maximal inhibition observed in heart membranes were about 25% and 35% for BNP and CNP respectively, however the inhibitions in brain striatal membranes were smaller (approximately 20%). The inhibition was dependent on the presence of guanine nucleotides and was attenuated by pertussis toxin treatment. In addition, CNP inhibited the stimulatory effect of isoproterenol on adenylyl cyclase, whereas CNP as well as BNP showed an additive effect with the inhibitory response of angiotensin II on adenylyl cyclase activity. When the combined effect of C-ANF4-23/BNP, C-ANF4-23/CNP and BNP/CNP at optimal concentrations was studied together on adenylyl cyclase activity, the percent inhibition remained the same for C-ANF4-23 and BNP or C-ANF4-23 and CNP, however, an additive inhibitory effect was observed for BNP and CNP. These results suggest that CNP and BNP like C-ANF4-23 interact with ANP-C receptors and result in the inhibition of adenylyl cyclase activity. On the other hand, CNP and BNP interact with the ANP-C receptor, however, the interaction may be different sites or there may be two subpopulations of ANP-C receptors specific for each of the peptides. These results indicate that BNP and CNP, like ANP and C-ANF4-23, inhibit the adenylyl cyclase/cAMP signal transduction system through an inhibitory guanine nucleotide regulatory protein, by interacting with ANP-C receptor subtypes.