Alterations in the genomic DNAs of six heterogeneous prostate carcinomas, as well as that of individual and histologically distinct foci within the tumors, were examined using restriction landmark genomic scanning, a method employing two-dimensional gel analysis of a large number of DNA fragments generated by digestion with highly specific endonucleases. Upon autoradiographic imaging, these fragments appear as spots of varying intensity and location specific for each sample. In our study, comparison of cancer DNAs against normal prostate DNA controls yielded alterations in at least 35 spots. Despite differences in the histological grading of tumors, 3 spots common to all tumor samples showed consistent amplification of intensity and 8 other common spots demonstrated consistent reduction of intensity when compared with control. In addition, spot alterations occurred between histologically identical foci isolated from within single tumors. We suggest that these spot changes detected in DNA profiles generated by restriction landmark genomic scanning reflect aberrations in as yet unidentified oncogenes and tumor suppressor genes and indicate that prostate cancer is not only histologically heterogeneous and multifocal but also genetically multicentric.