The 16 K subunit of the vacuolar H + -ATPase (ductin) has been suggested to also play a role in gap junction channels. Since mutated 16 K subunits have transforming ability when transfected into NIH3T3 cells and since aberrant gap junctional intercellular communication (GJIC) is a hallmark of cancer cells, we hypothesized that mutated 16 K subunits might transform these cells via alteration of GJIC. When GJIC was measured by the dye-transfer assay, NIH3T3 cells transfected with the mutant 16 K protein genes (deletion of the fourth transmembrane domain or a point mutation at codon 143 from glutamic acid to arginine) showed signi®cantly lower levels of GJIC than those transfected with the vector alone or with the wild-type 16 K subunit gene. GJIC levels of NIH3T3 cells transformed by v-ras and v-src were not signi®cantly decreased, suggesting that low GJIC levels are not necessarily the result of cell transformation per se. NIH3T3 cells express C643 as a major connexin gene. Although cells transfected with mutated 16 K subunits showed a level of C643 protein expression similar to non-transfectants, their C643 protein was localized aberrantly, i.e. intracytoplasmically. These results indicate that mutant 16 K subunits with transforming ability translocate C643 proteins, thus inhibiting GJIC of NIH3T3 cells.