The DLC-1 gene encoding a regulator of the Rho family of small GTPases is altered in breast, prostate, colon, and liver cancer and has several characteristics of a tumor suppressor gene. DLC-1 overexpression causes inhibition of in vitro growth of liver tumor cells and complete suppression of in vivo tumorigenicity of breast tumor cells. Inactivation and aberrant expression of DLC-1 in human hepatocellular carcinoma (HCC) is frequently associated with hemizygous and homozygous genomic deletion and promoter methylation. Since inactivation of tumor suppressor genes in cancer cells is also commonly associated with point mutation, we evaluated the incidence of mutation of the DLC-1 gene by PCR-SSCP in 17 primary HCC and 18 HCC cell lines. One missense mutation was detected at codon 991 of exon 12 (C-->T transition, Val-->Ile) in an HCC cell line. In addition, two types of polymorphisms were identified: a G-->T at codon 745 of exon 9, a T-->C at 17 bp downstream of exon 2. While the pathogenic relevance of the intronic polymorphism is not known, the low rate of mutation of the DLC-1 gene in HCC implies that genomic deletion and promoter methylation primarily account for the altered expression and tumor suppressive inactivation of the DLC-1 gene.