Studies of oxytocin-induced phosphorylation of myosin light chain (MLC), resulting in myometrial contraction, suggest that extracellular Ca2+ influx is involved in its signal transduction. To explore the possibility that intracellular Ca2+ mobilization by oxytocin may also contribute to MLC phosphorylation, we investigated the relative contributions of these Ca2+ sources to oxytocin signal transduction in myometrium of pregnant rat. In pregnant rat myometrium, oxytocin-induced Ca2+ influx occurs via an L-type voltage-dependent Ca2+ channel. Treatment with verapamil, an antagonist specific for these channels, significantly diminished MLC phosphorylation observed in response to oxytocin administration without affecting the release of Ca2+ from intracellular Ca2+ stores. Furthermore, oxytocin-induced MLC phosphorylation was not observed when extracellular Ca2+ was not present. Our results clearly indicate that extracellular Ca2+ influx, rather than release from Ca2+ storage sites, is essential for oxytocin-induced MLC phosphorylation. Copyright (C) 2001 John Wiley Sons, Ltd.