Hepatocellular carcinoma (HCC) is increasing worldwide and is the fifth main cause of cancer-related death. HCC develops on a preneoplastic organ, the cirrhotic liver. Therefore, chemoprevention could play a role in the therapy of HCC. We evaluated the preventive effects of pioglitazone, a peroxisome proliferator-activated receptor gamma agonist, on the induction of early carcinogenic events. We monitored pre-neoplastic foci induced by a two-stage initiation/promotion model of hepatocarcinogenesis in rats, using diethylnitrosamine and acetylaminofluorene. Pioglitazone treatment was initiated the day after the first diethylnitrosamine injection. By quantitative morphometry and Western blot, we showed that pioglitazone significantly decreases the size of pre-neoplastic foci. Analysis of proliferation and apoptosis, assessed by immunohistochemistry, demonstrated decreased proliferation but no effect on cell death in rats treated with pioglitazone. These events were associated with an increased expression of the cyclin-dependent kinase inhibitor p27(kip1), compared to the non treated group. In conclusion, pioglitazone inhibits early carcinogenic transformation in a two-step rat model. As pioglitazone has a low toxicity profile, we believe it would be interesting to evaluate its effect in chemoprevention of HCC in humans in a clinical setting.