Estradiol (E-2) accelerates egg transport by a nongenomic action, requiring activation of estrogen receptor ( ER) and successive cAMP and IP3 production in the rat oviduct. Furthermore, E-2 increases IP3 production in primary cultures of oviductal smooth muscle cells. As smooth muscle cells are the mechanical effectors for the accelerated oocyte transport induced by E-2 in the oviduct, herein we determined the mechanism by which E-2 increases IP3 in these cells. Inhibition of protein synthesis by Actinomycin D did not affect the E-2-induced IP3 increase, although this was blocked by the ER antagonist ICI182780 and the inhibitor of phospholipase C (PLC) ET-18-OCH3. Immunoelectron microscopy for ESR1 or ESR2 showed that these receptors were associated with the plasma membrane, indicating compatible localization with E-2 nongenomic actions in the smooth muscle cells. Furthermore, ESR1 but not ESR2 agonist mimicked the effect of E-2 on the IP3 level. Finally, E-2 stimulated the activity of a protein associated with the contractile tone, calcium/calmodulin-dependent protein kinase II (CaMKII), in the smooth muscle cells. We conclude that E-2 increases IP3 by a nongenomic action operated by ESR1 and that involves the activation of PLC in the smooth muscle cells of the rat oviduct. This E-2 effect is associated with CaMKII activation in the smooth muscle cells, suggesting that IP3 and CaMKII are involved in the contractile activity necessary to accelerate oviductal egg transport.