It can be difficult to distinguish between malignant and benign disease of the region of the head of the pancreas using conventional methods. K-ras and p53 alterations occur frequently in malignancies in this region and are therefore candidate tumour markers. To define the utility of these alterations in interpreting pancreatic head cytology, the present study investigated to what extent alterations in the carcinomas were detectable on cytology and whether the alterations found in the cytology came from the carcinomas. Fifty-seven consecutive pancreaticoduodenectomy resection specimens (52 with a malignancy and five without) and the ductal brush cytology specimens collected post-operatively from these resection specimens were compared for the presence of K-ras and p53 alterations. K-ras mutations were detected using the polymerase chain reaction (PCR) and allele-specific oligonucleotide hybridization, and p53 alterations using immunochemical staining for the p53 gene product. After discrepancy analysis, the results from the resection specimens and corresponding brush cytology specimens were identical in 88 per cent for the K-ras analysis and in 84 per cent for the p53 analysis. In two cases, K-ras mutations found in the brush cytology specimens were not derived from the carcinoma but from pancreatic ductal hyperplasias. Intratumour heterogeneity and sampling error were also identified as causes for discrepant results. The five resection specimens without a malignancy and the corresponding brush cytology specimens were negative for both genetic alterations. In conclusion, the detection of K-ras and p53 alterations in cells obtained from the pancreatic head region might be a valuable adjunct to conventional cytology for the diagnosis of malignancies in the pancreatic head region. However, intratumour heterogeneity, mucinous pancreatic duct hyperplasia harbouring K-ras mutations, and sampling error will hinder their diagnostic accuracy in routine clinical use.