In the epididymis, Cx43 forms gap junctions between principal and basal cells but not between adjacent principal cells. Cx30.3, 31.1, and 32 were identified in adult rat epididymis by RT-PCR, whereas Cx26 was present in young rats. Postnatal development studies indicate that Cx26 mRNA was detectable only in the caput-corpus region of the epididymis and that levels increased by fivefold during the first 4 wk postnatally, when epithelial cells differentiate, and decrease to nondetectable levels thereafter. Cx31.1 and Cx32 mRNA levels were low throughout the epididymis in young rats and began to increase in the second and third weeks postnatally, when Cx26 levels are decreasing. Both Cx26 and Cx32 were localized to the lateral plasma membranes between adjacent epithelial cells of the epididymis. Colocalization studies indicate that Cx26 and Cx32 exist either independently of one another or can colocalize along the lateral plasma membrane of epithelial cells in young rats or between principal cells in the adult rat epididymis. The presence of multiple connexins (Cxs) and their differential regulation suggest that these play different roles in epididymal development.