We have used subtractive hybridization to isolate cDNAs overexpressed in SK-PC-1 pancreas cancer cells. Forty-five independent clones corresponding to 11 genes were identified. Their expression in cultured pancreas cancer cells, normal pancreas tissue, and normal exocrine pancreas cultures was examined by Northern blotting. cDNA clones can be grouped into two broad categories: (1) those corresponding to genes expressed at high levels both in tumor cell lines and in primary cultures of normal pancreas, but not in normal tissue (i.e. thymosin beta4(3), cytokeratin 18, beta-actin, pyruvate kinase and mitochondrial genes); and (2) those corresponding to genes expressed at high levels in pancreas cancer cultures but not in normal pancreas tissue or cultured cells (i.e. tissue-type plasminogen activator and cathepsin H). The overexpression of these proteases in pancreas cancers suggests that they play a role in the aggressive biological behavior of this tumor.