The aim of the present study was to characterize the action of the neurotransmitter, NO, on rat myenteric neurons. A NO donor such as GEA 3162 (10^-4 mol·l^-1) induced an increase in the intracellular Ca²⁺ concentration as indicated by an increase in the fura-2 ratio in ganglia loaded with this Ca²⁺-sensitive fluorescent dye. The effect of GEA 3162 was strongly reduced in the absence of extracellular Ca²⁺ suggesting an influx of Ca²⁺ from the extracellular space evoked by NO. A similar nearly complete inhibition was observed in the presence of Ca²⁺ channel blockers such as Ni²⁺ (5·10^-4 mol·l^-1) or nifedipine (10^-6 mol·l^-1). Whole-cell patch-clamp recordings confirmed the activation of voltage-dependent Ca²⁺ channels, measured as inward current carried by Ba²⁺, by the NO donor. The peak Ba²⁺-carried inward current increased from -100 ± 19 pA to -185 ± 34 pA in the presence of sodium nitroprusside (10^-4 mol·l^-1). The consequence was a hyperpolarization of the membrane, which was blocked by intracellular Cs⁺, and thus most probably reflects the activation of Ca²⁺-dependent K⁺ channels. Furthermore, at least two subtypes of NO-synthases, the NOS-1 (neuronal form) and the NOS-3 (endothelial form), were found as transcripts in mRNA isolated from the rat myenteric ganglia. The expression of these NO-synthases was confirmed immunohistochemically. These observations suggest that NO does not only affect target organs such as smooth muscle cells in the gut, but has in addition profound effects on the enteric neurons themselves, the key players in the regulation of many gastrointestinal functions.