Mechanisms involved in the protective action of nitric oxide (NO) in insulin producing cells are a matter of debate. We have previously shown that pharmacological inhibition of c-Src cancels the antiapoptotic action of low and sustained concentrations of exogenous NO (Cell Signal 13:809-817, 2001). In this study, using insulin producing RINm5F cells that overexpress Src either permanently active (v-Src) or dominant negative (dn-Src) forms, we determine that this tyrosine kinase is the principal mediator of the protective action of NO. We also show that Src-directed activation of IRS-1, PI3K, Akt and Bad phosphorylation conform a substantial component of the survival route since pharmacological inhibition of PI3K and Akt cancelled the antiapototic effects of NO. Studies performed with the PKG inhibitor KT-5823 revealed that NO-dependent activation of c-Src/ IRS-1 is not affected by PKG activation. By contrast, Akt and Bad activation are partially dependent on PKG activation. Endogenous production of NO following overexpression of eNOS in RINm5F cells mimics the effects produced by generation of low amounts of NO from exogenous DETA/NO. In addition, we found that NO produces c-Src/PI3K- and PKG-dependent activation of Erk 1/2. The MEK inhibitor PD 98059 suppresses NO-dependent protection from DNA fragmentation induced by serum deprivation. The protective action of low and sustained concentration of NO is also observed in staurosporine and taxol-induced apoptosis. Finally, NO also protects isolated rat islets from DNA fragmentation induced by serum deprivation. These data strengthen the notion that NO production at physiological levels plays a role in protection from apoptosis in pancreatic -cells.