The insulin receptor mediates a proliferative response in certain transformed cells, but little is known about its function in ovarian cancer. We used human epithelial ovarian carcinoma cell lines and lifespan-extended normal ovarian surface epithelial (OSE) cells to examine ¹²⁵I-insulin binding and mitogenic responses to insulin. All cancer cell and OSE cultures specifically bound ¹²⁵I-insulin. Except for OV202, the carcinoma lines had elevated insulin binding compared with OSE cells. All carcinoma lines except OV202 expressed insulin receptor as detected by flow cytometry and increased ³H-thymidine incorporation or cell number in response to 0.1–10 nm insulin. Interestingly, similar concentrations of IGF-II also induced proliferation of the insulin-responsive cancer cell lines and displaced ¹²⁵I-insulin binding. Direct binding of ¹²⁵I-IGF-II to the insulin receptor was visualized by cross-linking and immunoprecipitation. Binding of IGF-II to the insulin receptor and a proliferative effect of insulin suggest the presence of insulin receptor isoform A. Real-time PCR analyses confirm that insulin receptor isoform A expression predominates over isoform B expression in the ovarian carcinoma cell lines. This report suggests that the insulin receptor may play a role in the regulation of ovarian cancer cell growth.