Tonic contraction of corpus cavernosum smooth muscle cells (SMCs) maintains the flaccid state of the penis, and relaxation is initiated by nitric oxide (NO), leading to erection. Our aim was to investigate the effect of NO on the smooth muscle cellular response to adrenergic stimulation in corpus cavernosum. Fura-2 fluorescence was used to record intracellular Ca²⁺ concentration ([Ca²⁺]_i) from freshly isolated SMCs from rat and human. Phenylephrine (PE) transiently elevated [Ca²⁺]_i, in the presence and absence of extracellular Ca²⁺, indicating release from intracellular stores. Whereas the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), with sildenafil citrate (SIL) caused no change in basal [Ca²⁺]_i, the PE-induced rise of [Ca²⁺]_i was reversibly inhibited by 27 ± 7 % (n = 21, p<0.005) in rat, and by 55 ± 15 % (n = 9, p<0.01) in human SMCs. SNAP and SIL also reduced the contractile response to PE. To investigate the mechanism, we applied mediators alone or in combination. The soluble guanylyl cyclase inhibitor, ODQ, reduced the effect of SNAP and SIL. Neither SIL, cGMP analogues, nor NO donors without SIL reduced the PE-induced rise of [Ca²⁺]_i. The combination of 8-bromo-cGMP with SNAP, however, reduced the Ca²⁺ peak by 42 ± 9 % (n = 22, p<0.01). Our results demonstrate that NO and cGMP act synergistically to reduce Ca²⁺ release from intracellular stores. Reduction of intracellular Ca²⁺ release may contribute to relaxation of the corpus cavernosum, leading to erection.