Monoclonal antibodies to human luteinizing hormone (hLH) were generated by a modified hybridoma technique. Out of forty hybrid cell lines that were shown to secrete antibodies reacting with hLH, LH35 and LH40 were further characterized biochemically and immunologically. LH35 was found to secrete immunoglobulin G1 antibody specific to the alpha-subunit of LH, whereas that of LH40 was beta-subunit specific. Association constants between the antibodies and LH were determined to be 2 X 10(8) and 1 X 10(9) M-1, respectively, by using competitive radioimmunoassay and Scatchard plots. Monoclonal antibodies from LH35 and LH40 were purified from the respective ascites fluids by ammonium sulfate fractionations and DEAE ion-exchange chromatography. The purified alpha-subunit-specific antibody of LH35 was immobilized on polystyrene balls (6 mm in diameter), whereas purified LH40 antibody was conjugated with horseradish peroxidase or labeled with iodine-125. Solid-phase radio- and enzyme immunoassays were designed to measure relatively low concentrations of LH (2-100 mIU/ml). The LH surge during the midcycles of women with normal menstrual cycles could easily be detected from daily urine or serum specimens by a 1-h assay procedure. It is proposed that this new LH immunoassay procedure can be routinely used for predicting ovulation of women with normal menstrual cycles.