Splenic lymphocytes of BALB/c mice immunized with a glycoprotein-enriched fraction of human ovarian adenocarcinoma were fused with the mouse myeloma cell line P3/NS1/1-Ag4 in the presence of polyethylene glycol (Mr 4,000). The hybrid cultures were screened in an indirect solid-phase radioimmunoassay for the production of relevant antibodies. Hybrids that produced antibodies which bound to the glycoprotein-enriched fractions of ovarian tumors but not to the similar fractions prepared from pooled normal ovary or sera were cloned twice by the limiting dilution method. Two such clones designated 4F4 and 7A10 were expanded in culture and also were grown in mice as ascitic tumors. The immunoglobulin isotype of the clones was of immunoglobulin G1 subclass with kappa light chains. Immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect the target antigen in 125I-labeled glycoprotein-enriched fractions of ovarian tumors. A single-chain Mr 48,000 peptide was identified by both clones 4F4 and 7A10. This antigen, which showed binding to concanavalin A-Sepharose, was designated gp48. Monoclonal antibodies against gp48 reacted significantly in radioimmunoassay to approximately 90% of human ovarian tumors and 60% of other tumors, both benign and malignant, but not to normal adult tissues or sera. Quantitative absorption analyses indicated that although the antigen was present in small amounts in some normal adult tissues such as cervix and intestine, it was present in much higher concentrations in most ovarian tumors, in some other tumors, and in fetal intestine and liver. Immunoperoxidase staining of formalin-fixed paraffin-embedded sections of solid ovarian adenocarcinomas revealed strong epithelial reactivity. Monoclonal antibodies to gp48 may be of value for the follow-up and immunotherapy of a variety of human tumors.