Pancreatic adenocarcinoma is among the most fatal of cancers, in part because of late diagnosis and a lack of effective therapies. Comprehensive studies are needed to better understand and address the cellular mechanisms and pathways of tumorigenesis. Serial analysis of gene expression was used to analyze gene expression profiles of pancreatic cancer cell lines, short-term cultures of normal pancreatic ductal epithelium, and primary pancreatic cancer tissue. A total of 294,920 tags representing 77,746 genes in 10 serial analysis of gene expression libraries were analyzed. A pancreatic cancer cell line (Hs766T) that exhibited a "normoid" profile of gene expression was identified. Several genes that may be involved in the fundamental nature of malignant changes in pancreatic ductal epithelium were suggested from those differentially and highly expressed in pancreatic cancer cells as compared with normal epithelium. Some overexpressed genes, such as S100A4, prostate stem cell antigen, carcinoembryonic antigen-related cell adhesion molecule 6, and mesothelin, suggest potential use as diagnostic markers. Others suggest potential novel therapeutic targets.