Increased myometrial expression of cyclooxygenase-2 (Cox-2) at term results from elevated local levels of inflammatory cytokines, and its inhibition provides a potential route for intervention in human pre-term labor. We have identified a role for atypical protein kinase C (PKC) isozymes in IL-1β-induced Cox-2 expression in human myometrial smooth muscle cells (HMSMC). The PKC inhibitor GF109203X (10 µM) inhibited IL-1β-induced Cox-2 protein and RNA expression, which were also reduced by MAPK and nuclear factor κB (NF-κB) inhibitors. GF109203X did not affect MAPK activities, and neither did it replicate the effect of p38 MAPK inhibition on Cox-2 mRNA stability, suggesting that PKC operates through an independent mechanism. The effect of GF109203X remained intact after depletion of conventional and novel PKC isozymes by phorbol ester pre-treatment. In contrast LY379196 (10 µM), which at micromolar concentrations inhibits all but atypical PKCs, did not affect Cox-2 expression. A peptide corresponding to the pseudosubstrate sequence of atypical PKCs blocked Cox-2 protein expression, whereas the sequence from conventional PKCs was ineffective. GF109203X did not affect NF-κB binding to nuclear proteins, but strongly reduced NF-κB-dependent transcription in luciferase reporter assays. Our findings indicate that IL-1β-induced Cox-2 expression in HMSMC in culture requires p38-MAPK-mediated mRNA stabilization and an independent activation of Cox-2 transcription which is dependent on the action of atypical PKCs, probably through direct stimulation of the transactivating activity of NF-κB. J. Cell. Physiol. 210: 637–643, 2007. © 2006 Wiley-Liss, Inc.