Abstract In rodents an action of estrogen is required for the initiation of implantation of the blastocyst into the endometrium of a progesteroneprimed uterus. Thus removal of endogenous estrogen, either directly by ovariectomy or indirectly by hypophysectomy, prevents implantation in the pregnant rat. In the present study, delayed implanting hypophysectomized progesterone-primed rats were used to test the efficacy of cyclic adenosine 3′,5′-monophosphate (cAMP), epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I), which are agents that have been shown to mimic some uterine actions of estradiol, to initiate implantation. In confirmation of previous studies, EGF injected into the uterine lumen plus intravenously was effective at initiating implantation in all animals. IGF-I showed no such activity in this model system. Cyclic AMP, increased via direct activation of adenylyl cyclase by forskolin, or administration of sodium dibutyryl cAMP, did not initiate implantation. However, a ligand for protein kinase C (PKC), phorbol 12-myristate 13-acetate, was effective in augmenting the action of intrauterine EGF, or forskolin, for initiation of implantation. A phorbol ester that does not activate PKC was ineffective. The results provide circumstantial evidence for the requirement of PKC activity in the implantation initiating action of estrogens.