The oxytocin receptor (OTR) mediates a wide spectrum of biological actions and is expressed in a large number of different tissues, including uterine, breast and lung tumors. To define more completely the intracellular signaling mechanisms linked to OTR activation, we have used a phospho-proteomics approach and have characterized changes in the phosphorylation states of intracellular proteins in response to OTR activation in OTR-expressing cell lines. Using a specific anti-phosphothreonine antibody, we observed several distinct changes in the threonine-phosphorylation patterns. The most prominent change involved dephosphorylation of a 95kD moiety. Purification by ion exchange chromatography combined with 1- and 2-dimensional PAGE followed by N terminal micro-sequence analysis revealed that the 95kD moiety corresponded to eukaryotic elongation factor 2 (eEF2). This protein is a key regulator of cellular protein synthesis and mediates, upon dephosphorylation, the translocation step of peptide chain elongation. Dose-response curves in myometrial cells expressing the endogenous OTR indicated a significant effect of OT on eEF2 dephosphorylation at 1 nM, a concentration close to the Kd of OT. Time course analysis indicates that the effect is rapid with a significant effect occurring at 5 min. To determine directly the effect of OT on protein synthesis, the incorporation of [³⁵S]Met into total protein was assessed. In myometrial cells, OTR activation led to significant 29% increase in total protein synthesis over a 2 h period. These findings establish a novel link between OTR activation and cellular protein synthesis and thus define a mechanism by which OT assumes a so far unrecognized, physiologically relevant trophic function.