We analyzed the expression of the retinoblastoma (Rb) gene in a group of ovarian neoplasms previously characterized for mutations in the p53 suppressor gene and the Ki-ras oncogene. Using immunohistochemical techniques, a total of 59 ovarian neoplasms spanning the histiologic spectrum from benign to malignant were examined for the expression of the Rb protein. All benign cystic adenomas and low malignant potential tumors exhibited normal expression of the Rb protein. Abnormalities in Rb protein staining were noted in 3 of 22 (14%) ovarian carcinomas. The staining patterns included tumors that were totally or focally negative for Rb protein. One tumor focally expressed Rb. This tumor demonstrated a direct juxtaposition of sections of Rb expressing and nonexpressing malignant epithelial cells. Two of the three tumors with abnormal Rb expression also had p53 mutations and staining on serial sections demonstrated that selected ovarian cancer cells possessed mutations in both oncogenes. These data suggest that the loss of Rb gene expression may play a role in the pathogenesis of a small number of invasive ovarian malignancies, but not in noninvasive ovarian neoplasms. © 1995 Academic Press. All rights reserved.