Previously, we demonstrated that nuclear factor-kappaB (NF-kappaB) mediates cytokine-induced hepatic inducible nitric oxide synthase (iNOS) expression. NF-kappaB activation is regulated by kinases and phosphatases whose function is only beginning to be understood. Therefore, experiments were performed to determine the role of protein phosphatases (PPase) in cytokine-induced iNOS expression. Hepatocytes were stimulated with cytokines in the presence or absence of tyrosine phosphatase inhibitors (pervanadate [PV], phenylarsine oxide [PAO]) and a serine-threonine phosphatase inhibitor (okadaic acid [OA]). Cytokines induced hepatocyte iNOS mRNA, protein, and NO2- production that was substantially decreased by the addition of the tyrosine phosphatase inhibitors (PAO and PV). The serine-threonine phosphatase inhibitor (OA) decreased NO release and protein levels in a concentration-dependent fashion; however, iNOS mRNA levels were not significantly reduced. Nuclear run-on experiments demonstrated that protein tyrosine phosphatases (PTPases) are required for iNOS transcription, while the serine-threonine phosphatase inhibitor (OA) had no effect on iNOS transcription. Electromobility shift assays (EMSAs) revealed that the tyrosine-phosphatase inhibitors blocked cytokine-induced NF-kappaB activation, while OA did not have a significant effect on NF-kappaB DNA binding activity. Therefore, tyrosine phosphatases are involved in the regulation of cytokine-induced activation of NF-kappaB, while serine-threonine phosphatases posttranscriptionally regulate iNOS translation. These results identify the regulatory role of specific protein phosphatases (PPases) in hepatic iNOS expression.