In order to explore nongenomic actions of estradiol (E₂) and progesterone (P₄) in the oviduct, we determined the effect of E₂ and P₄ on oviductal protein phosphorylation. Rats on Day 1 of the cycle (C1) or pregnancy (P1) were treated with E₂, P₄, or E₂ P₄, and 0.5 h or 2.5 h later their oviducts were incubated in medium with ³²P-orthophosphate for 2 h. Oviducts were homogenized and proteins were separated by SDS-PAGE. Following autoradiography, protein bands were quantitated by densitometry. The phosphorylation of some proteins was increased by hormonal treatments, exhibiting steroid specificity and different individual time courses. Possible mediation of the E₂ effect by mRNA synthesis or protein kinases A (PK-A) or C (PK-C) was then examined. Rats on C1 treated with E₂ also received an intrabursal (i.b.) injection of α-amanitin (Am), or the PK inhibitors H-89 or GF 109203X, and 0.5 h later their oviducts were incubated as above plus the corresponding inhibitors in the medium. Increased incorporation of ³²P into total oviductal protein induced by E₂ was unchanged by Am, whereas it was completely suppressed by PK inhibitors. Local administration of H-89 was utilized to determine whether or not E₂-induced egg transport acceleration requires protein phosphorylation. Rats on C1 or P1 were treated with E₂ s.c. and H-89 i.b. The number and distribution of eggs in the genital tract assessed 24 h later showed that H-89 blocked the E₂-induced oviductal egg loss in cyclic rats and had no effect in mated rats. It is concluded that E₂ and P₄ change the pattern of oviductal protein phosphorylation. Estradiol increases oviductal protein phosphorylation in cyclic rats due to a nongenomic action mediated by PK-A and PK-C. In the abscence of mating, this action is essential for its oviductal transport accelerating effect. Mating changes the mechanism of action of E₂ in the oviduct by waiving this nongenomic action as a requirement for E₂-induced embryo transport acceleration.