Cyclin D1/PRAD 1, a cell cycle-related gene mapped to chromosome 11q13, has been found to be amplified in some breast cancers and squamous cell carcinomas of the head and neck, and esophagus. In this study, overexpression of cyclin D1/PRAD1 gene was demonstrated immunohistochemically in 35 of 43 (81.3%) cases of human breast cancer, with a newly available anticyclin D antibody. Neither normal epithelial components nor glandular structures from samples of fibrocystic disease, were reactive. Amplification of the gene was detected in 4 of 23 (17%) cases by Southern analysis. Increased gene dosage does not seem to be the only mechanism that resulted in increased protein expression as detected by immunohistochemistry. Because the less differentiated high grade tumors exhibited a more intense nuclear stain and non-neoplastic epithelial components were not stained, the use of cyclin D1/PRAD1 has potential as a tumor marker.