Multiple lines of evidence indicate that inhibition of gap junction intercellular communication (GJIC) is a major carcinogenic process. Several reports suggest that activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) plays a key role in the disrupted GJIC by hydrogen peroxide, 12-O-tetradecanoylphorbol-13-acetate, and quinones in rat liver epithelial cells. Recently, we reported that p38 mitogen-activated protein kinase (MAPK) is also involved in the inhibition of GJIC by hydrogen peroxide in WB-F344 rat liver epithelial cells (WB cells). The present study investigated the role of ERK1/2 and p38 MAPK in H-ras-induced inhibition of GJIC in WB cells. H-ras induces complete inhibition of GJIC and unphosphorylation of connexin 43 (Cx43) in WB cells. SB203580, an inhibitor of p38, restored inhibition of GJIC and blocked unphosphorylation of Cx43 in H-ras-transformed WB cells. However, PD98059, an inhibitor of ERK1/2, had no effect. Our results suggest that the disruption of GJIC induced by H-ras may be strongly related to the unphosphorylation of Cx43 via the activation of p38 but not ERK1/2. Thus, p38 is a key signaling molecule for H-ras-induced inhibition of GJIC in WB cells.