Distinction between benign adrenal cortical proliferative lesions and adrenal cortical carcinoma has been approached by a combination of histological, immunohistochemical, and macroscopical parameters. Modern imaging studies allow detection of small adrenal cortical lesions that may be incorrectly diagnosed. Differentiation between benign and malignant tumors of the adrenal cortex was attempted by microdissection of nine cases of adrenal cortical hyperplasia, 10 cortical adenomas, and 18 adrenal cortical carcinomas with subsequent polymerase chain reaction (PCR) amplification for loss of heterozygosity (LOH) of five microsatellites of putative tumor suppressor gene loci: p53 gene (17p), the neuroblastoma candidate gene (1p), the p16 gene (9p), the von Hippel Lindau gene (3p), and the retinoblastoma gene (13q). None of the hyperplastic lesions or cortical adenomas showed LOH of any of the gene markers used. Conversely, genetic changes were observed in 61% (11 of 18) of the cases of carcinoma. Forty-four percent of the lesions showed LOH for p53 (7 of 16). LOH of 1p, 3p, and 9p were seen in 22%, 22%, and 26%, respectively. LOH of the retinoblastoma gene was seen in 80% or four of five of the informative cases studied. We conclude that LOH studies may be used to distinguish malignant from nonmalignant adrenal cortical proliferations. Relative infrequency of LOH in 3p may furthermore help to differentiate adrenal lesions from clear cell carcinomas of the kidney.