Atrial natriuretic peptide (ANP) exerts its hypotensive, natriuretic and diuretic effects, almost in part, through the activation of nitric oxide synthase (NOS). The aim was to investigate the natriuretic receptor type and the signaling cascade involved in NOS activation induced by ANP. Male Wistar rats were sacrificed and NOS activity was determined in kidney, aorta and heart with L-[U14C]-arginine, as substrate. ANP and cANP (4-23), a selective NPR-C ligand, increased NOS activity in all tissues. ANP induced a more marked activation in aorta and kidney than cANP (4-23), but no difference in atria NOS activation was observed. NOS activity induced by both peptides was blunted by nifedipine (L-type channel blocker) and calmidazolium (calmodulin antagonist) in heart and aorta. In kidney, nifedipine and calmidazolium abolished NOS activity stimulated by cANP (4-23) but only partially inhibited NOS activity elicited by ANP. Gi inhibition with pertussis toxin abolished NOS activity stimulated by ANP and cANP in atria but only partially inhibited the increased NOS activity induced by ANP and cANP in kidney, aorta and ventricle. Our results show that NPR-C receptor would mediate the activation of NOS by ANP in atria. In kidney, aorta and ventricle, NOS activation would also involve NPR-A and/or B. ANP would interact with NPR-C coupled via Gi to activation Ca2+ -dependent NOS.