The ability of estradiol benzoate (E2 B) and diethylstilbestrol (DES) to affect the formation and internalization of gap junctions was examined in several uterine cell types from hypophysectomized rats. Both myometrial and serosal cells respond to daily administration of E2 B or DES by increasing gap junction membrane in a dose-dependent fashion. The myometrial cell response arises from a zero base with gap junctions detected within 24 h of a single injection of 500 micrograms E2 B, while five daily injections of 5 micrograms E2 B were required to induce their formation at the lower dosage. Uterine serosal cells exhibit small macular gap junctions 60 days posthypophysectomy with E2 B stimulation leading to an increase in the number of macular gap junctions and the induction of annular gap junctions. Myometrial cell gap junctions were modulated by L-thyroxine and progesterone when administered in combination with E2 B but were without effect when administered alone. Combination of indomethacin with E2 B injections antagonized E2 B-stimulated junction growth in both myometrial and serosal cells, however, only serosal cells responded to exogenous prostaglandin (PG) injections, with PGE1 increasing and PGF2 alpha decreasing the number of serosal cell gap junctions. These studies support the assumption that the induction of gap junctions in uterine myometrium is hormone dependent.