The role of nitric oxide (NO) in the occurrence of intracellular calcium concentration ([Ca²⁺]_i) oscillations in pituitary GH₃ cells was evaluated studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and N-nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca²⁺]_i oscillations disappeared in 68% of spontaneously active cells whereas 41% of the quiescent cells showed [Ca²⁺]_i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO-donors NOC-18 and SNAP. NOC-18 was ineffective in the presence of the L-type voltage-dependent calcium channels (VDCC) blocker nimodipine (1µM) or in Ca²⁺-free media. Conversely, its effect was preserved when Ca²⁺ release from of the intracellular Ca²⁺ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the IP₃-receptors blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca²⁺]_i oscillations by determining Ca²⁺ influx. Patch clamp experiments excluded that NO was directly acting on VDCC while suggesting that it determined membrane depolarization due to the inhibition of voltage-gated potassium (VGK) channels. NOC-18 and SNAP caused, indeed, a decrease in the amplitude of slow-inactivating (I_DR) and of ether-a-go-go-related gene (ERG) hyperpolarization evoked deactivating K⁺ currents. Similar results were obtained when GH₃ cells were treated with L-arginine. In conclusion, the present study suggests that, in GH₃ cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca²⁺]_i oscillations through an inhibitory effect on I_DR and on I_ERG.