A new anticancer tripeptide, L-proline-m-bis (2-chloroethyl) amino-L-phenylalanyl-L-norvaline ethyl ester hydrochloride (MF13), was investigated for its activity and mechanism in human hepatocellular carcinoma (HCC) cell lines. MF13 showed antiproliferative activities in the panel of 7 human HCC cell lines with IC50 in the range of 0.08-2.32 microM. A significant blockade in the S-phase occurred in tumor cells 12 h after their exposure to MF13. The inactivated Rb (phosphorylated Rb, pRb), which is present in the S-phase, was increased within 6 h of treatment. Bcl-2 expression was without change in hepatocarcinoma cells treated with MF13; however, a significant increase of bax was observed, resulting in a decreased ratio of bcl-2/bax. Increased activity of caspase-9, -8 and -3 was detected in the MF13 treated cells, indicating an activated pathway of apoptosis by MF13. Morphological examination as well as DNA gel electrophoresis demonstrated a nuclear fragmentation and DNA degradation in the form of multiple-unit DNA ladder in MF13 treated tumor cells. MF13 alone at 10 mg/kg (i.p.) inhibited HepG2 tumor in nude mice by more than 94% in volume. Bel-7402 tumor originated from a Chinese patient with HCC exhibited a sensitivity to MF13 similar to HepG2 in vivo. Antitumor effect of MF13 in the nude mice bearing human hepatocarcinoma (Bel-7402 or HepG2) was stronger than mitomycin C as well as its precursor m-sarcolysin (p<0.01), and comparable with cyclophosphamide. We believe MF13 merits consideration for further investigation as an agent against human hepatocellular carcinoma.