The INK4alpha/ARF locus encodes p14(ARF) and p16(INK4alpha) , that function to arrest the cell cycle through the p53 and RB pathways, respectively. Genetic alterations of p14ARF and their relationship with p16(INK4a) or p53 inactivation have not been characterized in hepatocellular carcinoma (HCC). We examined 40 pairs of HCCs/noncancerous liver tissues for homozygous deletions (HD), methylation and mutations of the INK4a/ARF locus and for mutations of p53, and analyzed their clinicopathological correlation. p16(INK4a), p53 and p14(ARF) were inactivated in 62.5% (25 out of 40), 42.5% (17 out of 40) and 20% (8 out of 40) of HCCs, respectively. Inactivation of p14(ARF) was always associated with the concomitant inactivation of p16(INK4a) and occurred more frequently in hepatitis C virus (HCV)-associated HCC (p=0.042). Inactivation of p16INK4a) occurred more frequently in older patients (p=0.0027. The predominant mechanism of inactivation of p14(ARF) was homozygous deletion (7 out of 8), while that of p16(INK4a) was methylation (21 out of 25). Although statistically insignificant, genetic alterations of p14(ARF) tended to occur in tumors with wild-type p53. Genetic alterations of the INK4alpha/ARF locus could occur in small HCCs. In contrast, p53 mutations occurred more frequently in advanced HCCs (p=0.041). Inactivation of either p14(ARF)/p53 or p16(INK4a) occurred in 80% (32 out of 40) and concomitant disruption of both pathways occurred in 40% (16 out of 40) of HCCs, respectively. These results suggest that p14(ARF), p16(INK4a) and p53 are differentially disrupted through distinct molecular mechanisms at different stages in HCC and that p14(ARF) and p53 appear to function in the same tumor suppression pathway in HCC