Previous investigations have supported or indicated a stimulatory role of the insulin-like growth factor II gene (IGF2) in hepatocarcinogenesis. We have studied the transcript levels, promoter usage, and imprinting status of the ICF2 gene and its relationship to H19 in human hepatocellular carcinomas (HCCs) and liver tumor cell lines. The activity of the IGF2 promoter P1 was lost in about 70% of the cases (18 of 25). This is the most prominent abnormality regarding the IGF2 regulation in this study. Total IGF2 as well as promoter P3 transcription were up-regulated in a small group of the tumors. Twenty genetically informative cases were obtained from 26 cases, thus excluding the probability of loss of heterozygosity of the IGF2 gene. Among these, nine showed abnormal monoallelic expression of IGF2. One HCC and one HCC cell line proved loss of functional imprinting of IGF2. H19 and IGF2 were regulated in parallel, and expression levels were variable. Taken together, the disruption of the IGF2 promoter regulation, particularly the loss of P1 activity, is a common feature of human HCCs. The loss of P1 activity explains the frequent loss of biallelic IGF2 expression and may potentially be used as a diagnostic or monitoring marker for human HCC.