Expression of the gap junction proteins (connexins) in human breast epithelium was studied in vivo and in vitro. A panel of sequence-specific anti-peptide antibodies was used to examine four connexin (Cx) isoforms by indirect immunofluorescence labeling. Antibodies to Cx43 readily detected gap junctions between the basal cells in major ducts, but less so within lobular/alveolar structures. Cx26 immunoreactivity was less abundant in beast epithelium but was observed between the luminal cells in major ducts and to a lesser extent in lobular/alveolar structures. Ultrastructural studies of normal human breast showed gap junctions between basal cells in ducts and lobules, but not between luminal cells or between luminal and basal cells. Immunomagnetically separated luminal and basal cells were grown in vitro. Basal cells expressed Cx43 at cell-cell attachment points whereas luminal cells showed only small amounts of immunolabeling with the Cx26 antibody which was generally not associated with the cell borders. Microinjection of Lucifer yellow into cultured luminal or basal cells indicated that basal cells have high levels of gap junctional communication, but dye transfer between luminal cells was difficult to detect by transfer of Lucifer yellow. Western blot analysis of purified luminal and basal cells indicated the presence of mainly Cx43 in both cell types. Polymerase chain reaction analysis of breast mRNA identified message for CX43 and to a lesser extent for Cx26; Cx32 was not detected in human breast, although it was present in mouse mammary gland. mRNA extracted from cloned cultures of human luminal and basal cells contained message for Cx43 and Cx26 in both cell types.