Mouse hepatocytes immortalized with a temperature-sensitive allele of the SV40 large T-antigen (CHST8 cells) were found to lack the high expression of the gap junction proteins Cx26 and Cx32 that characterizes normal mouse hepatocytes, expressing instead Cx43 and Cx45 at minimal levels. In order to examine the growth suppressive function of Cx32 on hepatocytes, we transfected these CHST8 cells with human Cx32 complementary deoxyribonucleic acid and measured the growth rates at 33, 37, and 39° C. Expression of human Cx32 and its messenger ribonucleic acid in the stable cell lines was confirmed by immunocytochemistry and by Western and Northern blots analyses. Dye transfer following lucifer yellow injection into the transfectants was extensive; Cx32 channels displayed unitary conductances of about 70 pS and were moderately voltage sensitive. When cultured at 33 and 39° C, growth rates of both parental cells and transfectants were of the same level. When examined at 37° C, growth rate of the transfectant, which highly expressed Cx32 at the membranes, was significantly decreased compared to the parental cells. However, no changes in the expression of Cx32 protein in the transfectants were observed between 33 and 37° C. These results suggest that Cx32 expression could inhibit hepatocyte growth in vitro using the conditional immortalized cells. Cx32 transfectants using a conditional immortalized mouse hepatocyte may be useful for examining the mechanisms of growth and differentiation in hepatocytes by gap junction expression.