Secretoneurin enhances the adhesion and transendothelial migration properties of monocytes and is a part of the peptide family encoded by the secretogranin II gene. The expression of the secretogranin II gene is upregulated in senescent endothelium. The present study was designed to examine the effects of secretoneurin on endothelium-dependent responsiveness. Isometric tension was measured in rings (with or without endothelium) of porcine coronary arteries. Secretoneurin did not induce contraction of quiescent or contracted rings. In preparations contracted by U-46619, relaxation was observed with high concentrations of the peptide. This relaxation was endothelium dependent and reduced by the nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester (l-NAME). It was abolished when the preparations were incubated with l-NAME in combination with the cyclooxygenase inhibitor indomethacin. The relaxation was not affected by the combination of 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) and 6,12,19,20,25,26-hexahydro-5,27:13,18:21,24-trietheno-11,7-etheno-7H-dibenzo[b,m][1,5,12,16]tetraazacyclotricosine-5,13-diiumditrifluoroacetate hydrate (UCL 1684), which abrogates endothelium-dependent hyperpolarizations. These results indicate that secretoneurin acutely induces relaxation through the activation of endothelial nitric oxide synthase (eNOS) and cyclooxygenase, with nitric oxide playing the dominant role. Prolonged (24 h) incubation with physiological concentrations of secretoneurin enhanced the relaxations to bradykinin and to the calcium ionophore A-23187, but this difference was not observed in preparations incubated with l-NAME or the calmodulin antagonist calmidazolium. Under these conditions, the relaxation to sodium nitroprusside remained unchanged. Incubation with secretoneurin significantly augmented the expression of eNOS and calmodulin as well as the dimerization of eNOS in cultures of porcine coronary arterial endothelial cells. These observations suggest that secretoneurin not only acutely causes but also, upon prolonged exposure, enhances endothelium-dependent relaxations.