The Gadd45 family of proteins, which includes alpha, beta, and gamma isoforms, has recently been shown to play a role in the G2/M cell cycle checkpoint in response to DNA damage; however, the mechanisms by which Gadd45 proteins inhibit cell cycle control are not fully understood. Using immunohistochemical analysis, we found that protein expression of Gadd45gamma, but not Gadd45alpha, was down-regulated in hepatocellular carcinoma. We thus investigated possible mechanisms by which Gadd45alpha and Gadd45gamma might differentially induce G2/M arrest in the human hepatoma Hep-G2 cell line. Flow cytometric analysis revealed significant G2/M arrest in cells transfected with either Gadd45alpha or Gadd45gamma. Importantly, we found that expression of either Gadd45alpha or Gadd45gamma activated the P38 and JNK kinase pathways to induce G2/M arrest. Taken together, these findings suggest that the induction of G2/M arrest by Gadd45alpha or Gadd45gamma involves activation of two distinct signaling pathways in Hep-G2 hepatoma cell lines.