Conjugated eicosapentaenoic acid (cEPA) selectively inhibited the activities of mammalian DNA polymerases (pols) and human DNA topoisomerases (topos). cEPA inhibited the cell growth of two human leukemia cell lines, NALM-6, which is a p53-wild type, and HL-60, which is a p53-null mutant, with LD50 values of 37.5 and 12.5 microM, respectively. In both cell lines, cEPA arrested in the G1 phase, and increased cyclin E protein levels, indicating that it blocks the primary step of in vivo DNA replication by inhibiting the activity of replicative pols rather than topos. DNA replication-related proteins such as RPA70, ATR and phosphorylated-Chk1/2 were increased by cEPA treatment in the cell lines, suggesting that cEPA led to DNA replication fork stress inhibiting the activities of pols and topos, and the ATR-dependent DNA damage response pathway could respond to the inhibitor of DNA replication. The compound induced cell apoptosis through both p53-dependent and p53-independent pathways in cell lines NALM-6 and HL-60, respectively. These results suggested the therapeutic potential of cEPA as a leading anti-cancer compound that inhibited activities of pols and topos.