To assess the involvement of the TSLC cascade in hepatocarcinogenesis, we investigated the expression and DNA methylation patterns of the genes Tslc1 and Dal-1 in hepatocellular carcinomas (HCC) induced using N-nitrosodiethylamine (DEN) in rats. Six-week-old male F344 rats received a single intraperitoneal injection of DEN at a dose of 10 mg/kg body weight, followed by combined treatment with partial hepatectomy and colchicine to induce cell-cycle disturbance and a selection procedure consisting of 2-acetylaminofluorene and carbon tetrachloride. Total RNA was extracted from 10 HCC, and the expression levels of Tslc1 and Dal-1 were measured using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. Three of 10 HCC showed reduced expression of Tslc1, compared with normal liver tissues, but no changes in the expression level of Dal-1 were found. For DNA methylation analysis, bisulfite sequencing was performed. The 5' upstream region of Tslc1 was methylated in the three HCC in which its expression was reduced, but was unmethylated in normal liver tissue. Western blot analysis also revealed reduced expression of Tslc1 protein in the three HCC. These results suggest that alterations to the TSLC cascade might have a role in hepatocarcinogenesis using DEN in rats.