Gap junctions are characteristically increased in the myometrium during term and preterm delivery and are thought to be essential for the development of labor contractions. The expression of connexin-43 (Cx-43), the major myometrial gap junction protein, is increased during delivery (associated with an increase in the plasma estradiol/progesterone ratio) and after estradiol treatment of ovariectomized nonpregnant rats. However, Cx-43 is only 1 member of at least 16 proteins encoded by this family of gap junction genes. Using a RT-PCR method, we identified the presence of another member of this family, Cx-26, in laboring rat myometrium. The temporal expression pattern of Cx-26 was assessed using Northern and Western analyses. In contrast to Cx-43, whose expression is low throughout the pregnancy but increases immediately before the onset of labor (day 23), the expression of Cx-26 increased on day 17, reached maximal levels between days 19-21, and fell to low levels before the onset of labor. Treatment of pregnant rats with progesterone beginning on day 20 (which blocks both the increase in Cx-43 expression and the onset of labor) maintained the elevated expression of Cx-26. Induction of preterm labor in rats after ovariectomy on day 17 inhibited the normal preterm increase in Cx-26 transcripts. Progesterone treatment of these animals reversed the effects of ovariectomy. Immunofluorescence data identified Cx-26 antigen in the cell membranes of myometrial cells and in the luminal and glandular epithelium of the endometrium in the late pregnant (day 21) uterus. These data suggest that the role of gap junction formation in the myometrium in relation to the maintenance of pregnancy and the onset of labor is much more complex than previously recognized. Myometrial cell-cell communication is afforded by at least two different gap junction proteins, Cx-43 and Cx-26, that not only exhibit temporally distinct patterns of expression but are also subject to differential regulation.