To investigate the role of p16 I N K 4 protein absence in hepatocellular carcinoma progression, we examined p16 I N K 4 expression immunohistochemically in 81 primary and 23 metastatic lesions of hepatocellular carcinoma, in which retinoblastoma protein status had been determined. p16 I N K 4 protein was absent from 44% of the total of 104 tumors. The rate of p16 I N K 4 absence was twice as high in metastatic lesions (74%) compared with primary lesions (36%) (P=0.001). Loss of p16 I N K 4 and/or retinoblastoma protein was significantly associated with decreased tumor differentiation, vascular invasion and metastasis. In conclusion, p16 I N K 4 protein absence, alone and together with loss of retinoblastoma protein, contributes to hepatocellular carcinoma progression.