In the present study, competitive cDNA library screening (CCLS) and cDNA microarray analyses were employed to identify differentially expressed genes in methylnitrosourea-induced rat mammary adenocarcinomas. The preliminary screening of 100 000 plaques by CCLS identified 1217 clones with differential expression. Dot-blot analysis of the isolated clones verified differential expression in 471 distinct genes. Confirmation of these 471 genes was conducted by performing reverse transcription-polymerase chain reactions, and a total of 160 genes were confirmed after comparing six rat mammary adenocarcinomas and three normal rat mammary glands. Fifty-nine of these showed lower expression in the adenocarcinomas while the remaining 101 were overexpressed in the tumors. Employing a cDNA microarray containing 588 known genes revealed an additional 33 differentially expressed genes in these tumors. Importantly, most of the identified genes demonstrated relatively reproducible overexpression or underexpression in individual tumors. Many of the altered genes determined by cDNA microarray analysis were oncogenes, tumor suppressor genes, or genes involved in cell cycle control and apoptosis. CCLS identified many others not previously associated with mammary carcinogenesis, including a novel gene named RMT-7. Preliminary studies to determine the applicability of this gene expression approach for detecting potential biomarkers for cancer chemoprevention was evaluated in rat mammary tumors obtained from animals treated with vorozole, a potent aromatase inhibitor. When genes exhibiting differential expression as determined by CCLS or cDNA microarray analysis were examined in control and vorozole-treated tumors, expression of 19 genes was found to be modulated significantly in tumors treated with vorozole. Further investigations into these identified genes should contribute significantly to our understanding of the molecular mechanisms of rat mammary tumorigenesis. In addition, the identified genes may become useful targets for drug development and potential biomarkers for monitoring treatment and prevention of breast cancer in humans.