Subtractive hybridization, selecting for mRNAs expressed in normal human mammary epithelial cells (NMECs) but not in mammary tumor cell lines (TMECs), led to the cloning of the human gap junction gene connexin 26 (Cx26), identified by its sequence similarity to the rat gene. Two Cx26 transcripts derived from a single gene are expressed in NMECs but neither is expressed in a series of TMECs. Northern analysis using rat Cx probes showed that Cx43 mRNA is also expressed in the normal cells, but not in the tumor lines examined. Connexin genes Cx31.1, Cx32, Cx33, Cx37, and Cx40 are not expressed in either normal cells or the tumor lines examined. In cell-cell communication studies, the normal cells transferred Lucifer yellow, while tumor cells failed to show dye transfer. Both Cx26 and Cx43 proteins were immunolocalized to membrane sites in normal cells but were not found in tumor cells. Further analysis demonstrated that Cx26 is a cell-cycle regulated gene expressed at a moderate level during G1 and S, and strongly up-regulated in late S and G2, as shown with lovastatin-synchronized NMECs. Cx43, on the contrary is constitutively expressed at a uniform low level throughout the cell cycle. Treatment of normal and tumor cells with a series of drugs: 5dB-cAMP, retinoic acid, okadaic acid, estradiol, or TGFb had no connexin-inducing effect in tumor cells. However, PMA induced re-expression of the two Cx26 transcripts but not of Cx43 in several TMECs. Thus Cx26 and Cx43 are both downregulated in tumor cells but respond differentially to some signals. Modulation of gap-junctional activity by drug therapy may have useful clinical applications in cancer.