Pancreatic cancer is the deadliest of cancers, and effective diagnostic and therapeutic strategies are lacking. Global gene expression profiling holds promise for improved diagnosis and treatment. Knowledge of the location and timing of gene overexpression and the function of these genes, including their effects on signaling pathways, is important. These data may be used to develop histologic and serum biomarkers as well as to develop immunotherapeutic, molecular targeting, and gene therapy strategies. We have compiled a list of overexpressed genes in pancreatic cancer for which overexpression was confirmed by reverse transcriptase polymerase chain reaction, immunohistochemistry, and/or in situ hybridization following initial identification by global gene expression profiling. The techniques used in the determination of overexpression, problems encountered in global gene expression profiling, and the diagnostic and therapeutic implications of overexpression are discussed. The S100 gene family, mesothelin, prostate stem cell antigen, and 14-3-3 sigma, may have important clinical implications in pancreatic cancer diagnosis and treatment.