A higher prevalence of epigenetic inactivation of tumor suppressor genes has been reported in cancer cell line populations compared to primary cancer populations. Cancer-related genes are commonly methylated in cancer cell lines but it is not known the extent to which tumor suppressor genes may be artificially methylated in vitro. We therefore examined 10 pancreatic cancer cell lines and corresponding primary tumors for aberrant DNA methylation of promoter CpG islands of eight genes and seven CpG islands. Using methylation-specific PCR (MSP), methylation was not detected at any of the 15 CpG islands in 15 normal pancreata or in an immortalized normal pancreatic duct epithelial (HPDE) cell line. Of 150 loci examined, 49 loci were methylated in both primary carcinomas and their corresponding cell lines, 95 loci were not methylated in either cell lines or their corresponding primary carcinomas. There were four loci methylated only in cell lines while another two loci were methylated only in primary carcinomas. Overall, the methylation status of primary carcinomas and their cell lines were concordant in 96% of cases (144 of 150) (J statistic; J=0.92, P<0.0001). We conclude that most of the DNA methylation of tumor suppressor genes observed in cancer cell lines is present in the primary carcinomas from which they were derived.