Regulation of prostaglandin endoperoxide synthase-2 (PGHS-2) mRNA levels by serine-threonine phosphatases was examined in murine fibrosarcoma methylcholanthrene-101 cells. Okadaic acid (OA), a serine-threonine phosphatase inhibitor, induced PGE2 production and a significant increase in PGHS-2 immunoreactive protein. A specific PGHS-2 inhibitor, N-(2-cyclohexyloxy-4-nitrophenyl) methanesulphonamide, completely abolished the OA-mediated increase in PGE2 production, which suggests that the PGE2 formed in response to OA was derived from PGHS-2. OA-mediated PGHS-2 mRNA accumulation was observed at 1 hr, remained elevated for 24 hr and was blocked by actinomycin D, which indicates that OA increases PGHS-2 gene transcription. A significant post-transcriptional mechanism also contributed to the increased PGHS-2 mRNA accumulation, because the mRNA half-life was approximately 4 to 5 h in OA-stimulated cells. Tumor necrosis factor-alpha, but not OA, activated transcription factor nuclear factor-kappaB in methylcholanthrene-101 cells, as demonstrated by translocation of the nuclear factor-kappaB complex to the nucleus and disappearance of the cytoplasmic inhibitory protein, IkappaB-alpha. We conclude that inhibition of serine-threonine phosphatases contributes to the up-regulation of PGHS-2 expression in an NF-kappaB-independent manner.