The nonapeptide oxytocin mediates a wide spectrum of biological action many of them related to reproduction. Recently, we have shown that oxytocin exerts a trophic effect on uterine smooth muscle cells and induces dephosphorylation, and thus activation, of the translation elongation factor eEF2 (eukaryotic elongation factor 2). The present study was designed to elucidate the mechanisms underlying this novel action of oxytocin in the well characterized human myometrial cell line hTERT-C3. Pathways known to induce eEF2 dephosphorylation are mTOR and the MAP kinases ERK1/2 and p38. Using a panel of chemical inhibitors of specific signaling pathways, we determined that none of these pathways played a role in oxytocin-mediated eEF2 dephosphorylation. Because the oxytocin receptor is a G protein-coupled receptor linked to Gq, we tested the possibility that this oxytocin action was mediated via protein kinase C (PKC). PKC activity was blocked by application of the general PKC chemical inhibitor Go6983 or by incubation with the cell-permeable PKC inhibitor peptide myr-psi PKC. With either approach, the effect of oxytocin on eEF2 dephosphorylation was suppressed, indicating that the PKC pathway is essential for this oxytocin action. Consistent with this idea, we also found that direct stimulation of PKC with the phorbol ester PMA induced eEF2 dephosphorylation. Moreover, we observed that the stimulatory effect of oxytocin on [³⁵S]methionine incorporation into nascent proteins was blocked by PKC inhibition. Overall, these results define a novel hormonal signaling pathway that leads to eEF2 dephosphorylation and activation of protein synthesis.