We have previously shown that pretreatment of A-10 vascular smooth muscle cells (VSMC) with angiotensin II (Ang II) attenuated atrial natriuretic peptide receptor-C (ANP-C)-mediated inhibition of adenylyl cyclase without altering [ 125I]ANP binding. In the present studies, we have investigated the modulation of ANP-C receptor signaling by arginine-vasopressin (AVP). Pretreatment of A-10 VSMC with AVP for 24 h resulted in a reduction in ANP receptor binding activity by about 50% ( B max; control cells, 22.9 ± 2.5 fmol/mg protein, AVP-treated cells, 11.4 ± 1.2 fmol/mg protein). In addition, the expression of ANP-C receptor as determined by immunoblotting was also decreased by about 50% by AVP treatment, which was prevented by GF109203X, an inhibitor of protein kinase C (PKC). The decreased expression of ANP-C receptor was reflected in an attenuation of ANP-C receptor-mediated inhibition of adenylyl cyclase. C-ANP 4–23 [des(Gln 18,Ser 19,Gln 20,Leu 21,Gly 22)ANP 4–23–NH 2], a ring deleted peptide of ANP that interacts specifically with ANP-C receptor, inhibited adenylyl cyclase activity by about 30% in control cells, which was completely attenuated in AVP-treated cells. This attenuated inhibition was significantly restored by GF 109203X. In addition, AVP treatment augmented the levels of Giα-2 and Giα-3 proteins; however, the Gi functions were completely attenuated. The increased expression of Giα proteins induced by AVP was inhibited by GF109203X as well as by actinomycin D treatments. In addition, AVP treatment also enhanced the expression of Gsα protein and Gsα-mediated stimulation of adenylyl cyclase by GTPγS, N-ethylcarboxamide adenosine (NECA), and forskolin (FSK), whereas the levels of Gβ were not altered by AVP treatment. These results indicate that AVP-induced PKC signaling may be responsible for the down-regulation of ANP-C receptor that results in the attenuation of C-ANP 4–23-mediated inhibition of adenylyl cyclase activity, and suggest a cross-talk between vasopressin V 1 and ANP-C receptor-mediated signaling pathways.