Myometrial tissues from pregnant and nonpregnant mature and immature rats were examined for the presence of gap junctions by thin section and freeze-fracture microscopy before and after incubation in vitro. Gap junctions were not present in any tissues fixed in situ or shortly after removal from animals. The junctions were present in tissues incubated for 2 h in vitro, and the number and size of the junctions increased with increasing incubation times up to 48 h. Tissues from immature rats incubated in vitro formed gap junctions in much reduced numbers as compared to tissues from pregnant or nonpregnant mature animals. Injection of immature animals with estrogen with or without progesterone, but not with progesterone alone, increased the number of gap junctions found in tissues when incubated in vitro. In vitro treatment of tissues from immature animals with estrogen also stimulated the formation of gap junctions. Progesterone treatment in vitro had no effect on gap junction formation in any myometrial tissues. However, progesterone added in vitro in the presence of estrogen decreased the number of gap junctions as compared to the numbers formed with addition of estrogen alone in treated tissues from immature or pregnant animals. The addition of indomethacin 5,8,11,14-eicosatetraynoic- acid and arachidonic acid partially inhibited the formation of gap junctions in tissues from pregnant animals incubated in vitro. A stable endoperoxide of PGH2 and arachidonic acid, but not PGE1, PGE2, PGF(2α), or thromboxane β2, overcame the inhibition of gap junction formation produced by indomethacin. These results are consistent with the hypothesis that steroid hormones and prostaglandin interact to modulate gap junction formation in the myometrium.