The exposure of rat mesangial cells to cytokines promoted activation of the p42/p44 mitogen-activated protein kinase (MAPK). We identified a rapid and delayed phase of MAPK activation with distinctive activity increases at 5 to 15 min and 15 to 24 h. Rapid and late MAPK activation were attenuated by the redox-modulating agent N-acetylcysteine. Specifically, late-phase activation coincided with endogenous nitric oxide (NO) generation and in turn was suppressed by the NO synthase-blocking compounds diphenyliodonium or nitroarginine methyl ester. By using NO-liberating agents such as S-nitrosoglutathione and 3-morpholinosydnonimine, we investigated intermediary signaling elements of NO in promoting MAPK activation. Early and transient activation at 5 min was suppressed by the soluble guanylyl cyclase-blocking agent 1H-(1,2,4)-oxdiazolo-(4,3-alpha)-6-bromoquinoxazin-1-one (NS 2028) and, moreover, was mimicked by the lipophilic cyclic GMP (cGMP) analogue 8-bromo-cGMP. In contrast, NO-mediated activation achieved within hours was unrelated to cGMP signaling. Late and persistent MAPK activation, induced by NO donors or endogenously generated NO, was found in association with inhibition of phosphatase activity. In vitro dephosphorylation of activated and immunoprecipitated p42/p44 by cytosolic phosphatases was sensitive to the readdition of NO and was found to be inhibited in cytosol of S-nitrosoglutathione-stimulated cells. Also, cells that had been exposed to cytokines for 24 h revealed a blocked phosphatase activity, which was successfully attenuated by the NO synthase inhibitor nitroarginine methyl ester and, therefore, was NO mediated. Conclusively, NO affects p42/p44 MAPK in rat mesangial cells twofold: rapid activation is cGMP mediated, whereas late activation is transmitted via inhibition of tyrosine dephosphorylation.