Regulators of G-protein signaling (RGS proteins) interact with G_q and G_i, and accelerate GTPase activity. These proteins have been characterized only within the past few years, so our understanding of their importance is still preliminary. We examined the effect of oxytocin on RGS2 mRNA expression to help determine the role of RGS proteins in oxytocin signaling in human myometrial cells in primary culture. Oxytocin increased RGS2 mRNA concentration maximally by 1 or 2 h, in a dose-dependent and agonist-specific manner. RGS2 mRNA levels were also elevated by treatment with calcium ionophore, phorbol ester, or forskolin. Oxytocin's effects were completely inhibited by an intracellular calcium chelator, and partially blocked by a protein kinase C (PKC) inhibitor, indicating that intracellular Ca²⁺ concentration is the primary signal for oxytocin elevation of RGS2 mRNA levels. Use of pharmacological inhibitors indicated that part of oxytocin-stimulated RGS2 mRNA expression is mediated by Gi/tyrosine kinase activities. Although oxytocin does not stimulate increases in intracellular cAMP concentration, agents that elevate intracellular cAMP concentrations and cause myometrial relaxation may possibly cause heterologous desensitization to oxytocin via RGS2 expression. These results suggest that RGS2 may be important in regulating the myometrial response to oxytocin.