We have developed a modified form of suppression subtractive hybridization (SSH) that allows multiple specimens of distinct phenotypic groups to be compared for consistent differences in gene expression. We applied this system to identify genes that were expressed in normal rat ovarian surface epithelial (ROSE) cells but whose expression was lost/downregulated in four independently transformed rat ovarian cancer cell lines. Northern blot analysis using 14 of 28 nonredundant cDNA fragments from this difference library showed that the mRNA transcripts were present in normal ROSE cells but lost or markedly downregulated in four related transformed cell lines. Of particular interest, cellular retinol-binding protein 1 (CRBP1) and retinol-binding protein (RBP), two genes whose products are involved in retinol transport and metabolism, were found to be downregulated in this ovarian cancer model system. To determine if this change had relevance to human ovarian cancer, we evaluated a series of human ovarian cancer cell lines and a limited number of frozen human ovarian tumors and found lost or decreased expression of CRBP1 and RBP relative to expression in human ovarian surface epithelial (HOSE) cells. We hypothesize that the loss of CRBP1 and RBP expression disrupts retinol metabolism and retinoic acid production, which may facilitate the occurrence of genetic damage leading to the malignant transformation of the ovarian surface epithelium, the cells from which ovarian cancer arises.