We investigated the effect of desferrioxamine (DFO), an iron chelator, on the DNA synthesis and the cell cycle of cultured hepatoma cells. Using Hep 3B cells as the hepatoma cell lines, DNA synthesis was measured by [³H] thymidine incorporation, and the cell cycle analysis was performed by flow cytometry including bivariate DNA/BrdU analysis. [³H] thymidine uptake was decreased by DFO in a dose dependent manner. The proportion of S phase cells increased and that of G0/G1 phase cells decreased after the addition of DFO in the culture media in a dose dependent manner up to 20 µg/ml of DFO. The S phase duration of the exponentially proliferating Hep 3B cells was 9.9 hours when cultured without DFO, but it was markedly prolonged (54.1 hours) after the addition of 20 µg/ml of DFO. After removal of DFO from the culture media following 24 hours of incubation with 20 µg/ml of DFO, a sequential increase from early through mid and late-S to G2/M phase was observed. In conclusion, the antiproliferative effect of DFO on cultured human hepatoma cell lines was caused by the inhibition of DNA synthesis which was related to a block in the early-mid S interface or mid S phase of the cell cycle.