The effect of linoleic acid (LA) on gap-junction permeability, connexin 43 mRNA level, protein level, and phosphorylation, and the numbers of gap-junctional membrane plaques were studied in the rat liver epithelial cell line WB-F344 to determine whether changes in these parameters correlated with the enhanced cell growth and the inhibition of gap-junction function. When cultured in a medium with low serum (1%), these cells exhibited a slower growth rate than in the high serum medium (7%). Addition of linoleic acid (0.01–3 mg/ml) to the low serum medium increased the growth rate and inhibited gap junctional intercellular communication (GJIC) in a dose-dependent manner. In a comparison of short-term and long-term treatments with LA, GJIC in short-term treated (1 h) WB cells was inhibited at 3 mg/ml LA but readily recovered by washing and removing LA from cells, whereas GJIC in long-term treated (6 days) WB cells did not recover by washing and removing LA from WB cells. Western blot analysis of connexin 43 showed that a short-term incubation with linoleic acid increased the relative amount of unphosphorylated connexin 43 protein, but a long-term incubation with linoleic acid decreased the amount of unphosphorylated connexin 43 protein and increased the relative amount of hyperphosphorylated connexin 43 protein. Connexin 43 and p53 mRNA levels decreased in a time- and dose-dependent manner in linoleic acid-treated cells. These results suggest that growth stimulation and gap junctional intercellular communication inhibition of rat liver epithelial cells by linoleic acid may be mediated in part through modulation of p53 expression and function.