In a study for the identification of genomic alterations in pancreatic cancer, representational difference analysis was used and led to the isolation of 2 distinct fragments, deleted on the Y chromosome in the xenografted tumor DNA of a male patient with an adenocarcinoma of the pancreas. Loss of Y chromosomal material was further studied in 11 pancreatic cancer cell lines of male origin, using PCR amplification of 5 sequence tagged sites (STSs) distributed along the Y chromosome; 8/11 cell lines exhibited a complete loss of the Y chromosome and 3 had deletions. To examine the status of the Y chromosome in situ, interphase FISH analysis was performed on paraffin sections from pancreatic carcinoma (n=7) and chronic pancreatitis (n=7) tissues, and the loss of Y-chromosomal STS-markers was studied in 6 xenograft tumors obtained from male pancreatic cancer patients. This analysis revealed that a loss of the Y chromosome occurs in vivo in primary pancreatic tumor cells, whereas the Y chromosome was intact in chronic pancreatitis. Our data suggest that loss of Y is a frequent event occurring in male pancreatic tumors. Although there is no evidence for a functional implication of Y chromosome loss, it effectively differentiates between a malignant and a benign condition as e.g. chronic pancreatitis. Thus, this genetic alteration may be of diagnostic use.