The mechanisms by which oxytocin (OT) stimulates extracellular signal-regulated kinase 1/2 (ERK1/2) are only partially understood. OT receptor (OTR) signals predominantly through G_q, but ERK1/2 phosphorylation (ERK1/2-P) in PHM1 myometrial cells was not eliminated by inhibition of downstream effectors such as PLC or PKC. Inconsistent with a G_i-coupled response, pertussis toxin inhibition of OT-induced ERK1/2-P was reversed by the protein kinase A inhibitors Rp-cAMPS and KT5720. Consistent with an inhibitory role for protein kinase A, pertussis toxin pretreatment raised cellular cAMP and CPT-cAMP inhibited OT-induced ERK1/2-P. Attenuation of the OT response by the G scavenger ARK-ct implicated a G-mediated pathway. In both COSM6 cells overexpressing OTR (OTR-COSM6) and in PHM1 cells, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 markedly reduced OT-induced ERK1/2-P, whereas the platelet derived growth factor (PDGFR) tyrosine kinase inhibitor AG1296 had no effect. Furthermore, OT increased EGFR tyrosine phosphorylation in OTR-COSM6 cells, which was inhibited by AG1478 or EGTA plus thapsigargin pretreatment. AG1478 did not affect IP₃ production by OT or PKC-stimulated ERK1/2-P but completely blocked ionomycin-induced ERK1/2-P and EGFR tyrosine phosphorylation. In both OTR-COSM6 and PHM1 cells, EGTA reduced OT-stimulated ERK1/2-P; no ERK1/2-P was observed when intracellular calcium increases were blocked by pretreatment with thapsigargin plus EGTA. These data are consistent with activation of a G-mediated pathway as a consequence of G_q activation in myometrium and OTR-COSM6 cells that results in increased ERK1/2-P. This pathway involves both EGFR activation and an influence of calcium.