This investigation examined the role of estrogen receptor (ER) on the stimulatory effect of estradiol (E₂) on protein phosphorylation in the oviduct as well as on E₂-induced acceleration of oviductal oocyte transport in cyclic rats. Estrous rats were injected with E₂ s.c. and with the ER antagonist ICI 182 780 intrabursally (i.b.), and 6 h later, oviducts were excised and protein phosphorylation was determined by Western blot analysis. ICI 182 780 inhibited the E₂-induced phosphorylation of some oviductal proteins. Other estrous rats were treated with E₂ s.c. and ICI 182 780 i.b. The number of eggs in the oviduct, assessed 24 h later, showed that ICI 182 780 blocked the E₂-induced egg transport acceleration. The possible involvement of adenylyl cyclase, protein kinase A (PK-A), protein kinase C (PK-C), or tyrosine kinases on egg transport acceleration induced by E₂ was then examined. Selective inhibitors of adenylyl cyclase or PK-A inhibited the E₂-induced egg transport acceleration, whereas PK-C or tyrosine kinase inhibitors had no effect. Furthermore, forskolin, an adenylyl cyclase activator, mimicked the effect of E₂ on ovum transport and E₂ increased the level of cAMP in the oviduct of cycling rats. Finally, we measured PK-A activity in vitro in the presence of E₂ or E₂-ER complex. Activity of PK-A in the presence of E₂ or E₂-ER was similar to PK-A alone, showing that E₂ or E₂-ER did not directly activate PK-A. We conclude that the nongenomic pathway by which E₂ accelerates oviductal egg transport in the rat requires absolute participation of ER and cAMP and partial participation of PK-A signaling pathways in the oviduct.