We investigated the expression of gap junctions in virgin, pregnant, lactating, and involuting mouse mammary gland epithelium with a panel of sequence-specific antibodies to connexins 26, 32, 40 and 43. Indirect immunofluorescence labeling of frozen sections of mammary gland showed that connexin26 was the major connexin in mammary epithelium. Connexins 43, 40, and 32 were not detected. Connexin26 was not detected in the mammary epithelium of virgin mice but was increasingly expressed during pregnancy. At Day 4 of pregnancy, when the mammary gland was composed almost exclusively of ducts, low levels of labeling were detected in the duct epithelium. As pregnancy progressed, the level of labeling with antibodies to connexin26 increased in quantity and intensity. At Day 12, when developing lobules were present, immunolabeling for connexin26 was detected surrounding the developing lumina, which on Day 19 were distended with milk. Labeling of mammary gland reached a maximum on Day 24 (5 days' lactation) but within 24 hr of removal of the litter on Day 28, connexin26 labeling was greatly diminished. No further change in labeling intensity with the antibodies to connexins was detected throughout involution. Double immunofluorescence labeling of 5-day lactating mammary gland with antibodies to connexin26 and anti-keratin 14 or -keratin 19 indicated that the majority of gap junctions detected by this analysis were within the luminal cell population. Western blot analysis of a lactating mammary gland (Day 24) confirmed the absence or low level of expression of connexins 32 and 43, as seen in the immunofluorescence studies, and showed that connexin26 was a dominant antigen expressed in lactating mammary gland epithelium.