During pregnancy, the integrity of the mammary gland epithelium must be maintained by the junctional nexus formed by gap, adherens, and tight junctions for proper alveologenesis. We have previously shown that mammary glands from Gja1^Jrt/ mice, harboring a mutation in the Gja1 gene encoding connexin (Cx) 43, have reduced Cx43 gap junction function and are unable to release milk to the pups. We hypothesized that low levels of Cx43 in the mammary gland may also affect other mammary gland connexins, tight and adherens junction proteins, and alveolar architecture, thus contributing to the lactation defect. Surprisingly, the levels and/or localization of Cx26 (Gjb2), Cx30 (Gjb6), Cx32 (Gjb1), E-cadherin, P-cadherin, beta-catenin, claudin-1, and occludin were all unaffected by the decreased levels of Cx43 at parturition, suggesting no cross-talk to other connexins or junctional proteins in mutant mice mammary glands. Consistently, in mammary gland myoepithelial primary cultures or in Normal Rat Kidney (NRK) cells, tight and adherens junction protein levels and localization were unaffected by the expression of Cx43 mutants. However, whereas the architecture of the alveolar and ductal structures appeared to be normal in mutant mice, the presence of milk and cytoplasmic lipid droplets were frequently observed within the epithelium itself, suggesting a defect in the secretory process. Overall, these results suggest that proteins associated with tight and adherens junctions are unaffected by greatly reduced Cx43 gap junctions in the mammary gland epithelium, and they further suggest that the mammary gland defect in Cx43 mutant mice may extend to impaired milk secretion.