K-ras mutations at codon 12 have been detected in almost all pancreatic adenocarcinomas by highly sensitive assays. We reassessed the K-ras mutation status by direct polymerase chain reaction (PCR) and sequencing from tissue microdissection without DNA extraction in 10 pancreatic adenocarcinomas, and also assessed the K-ras and DPC4 genes in nine pancreatic cancer cell lines. Eight pancreatic adenocarcinomas were found to harbor K-ras mutations at codon 12 of either GTT or GAT, five of which were inferred to harbor amplified mutant alleles. Mutations at the sites other than codon 12 were found in seven of 70 clones (seven of 9,380 bases) by the TA cloning analysis, suggesting that artifactual mutations at the first or second base of codon 12 before and during PCR could occur at a frequency of approximately 10(-3), enough for highly sensitive assays to detect. Two cell lines without K-ras mutations at codon 12 were found to have homozygous deletions at the DPC4 gene. Thus the K-ras mutation status was demonstrated to be correctly determined by just direct sequencing from tissue microdissection. All possible mutations or multiple mutations at K-ras codon 12 that have been reported in pancreatic adenocarcinomas might include artifacts or mutations without a selective advantage. In addition, we must be very cautious about contamination.