Monoclonal antibodies (mabs) to human (h) FSH were utilized to probe epitopes of the beta-subunit of hFSH (hFSH beta). These mabs had an average approximate affinity constant (Ka) of 10(8) M-1 for hFSH beta and 10(7) M-1 for heterodimeric hFSH. Hormone specificity of mabs for hFSH beta was demonstrated by a lack of cross-reactivity with hCG alpha, FSH alpha, or LH alpha. Epitope specificity of each mab was initially assessed by determining whether solid phase mab could bind to [125I]hFSH already bound to mabs in liquid phase. In addition, it was determined whether [125I]mab could bind to hFSH already bound to solid-phase mabs. Both epitope cross-matching protocols indicated that all mabs bound to the same epitopes on hFSH beta. Next, synthetic peptides corresponding to the sequence of hFSH beta were used in an enzyme-linked immunosorbent assay to map this epitope. All mabs bound to peptides 7-19, 1-20, 33-53 and 66-85 but did not bind or bound weakly to peptides 81-100, 95-103, and 103-110. Titration experiments were performed using different concentrations of peptide (0.3-41 nmol) and one mab 3G3 (500 ng-25 ng) in the enzyme-linked immunosorbent assay. The product of the lowest mass of both peptide and antibody which gave a positive result was used to rank the peptides for their binding with mab 3G3. Peptides were ranked in the following descending order of potency: 33-53, 49-67, 66-85 much greater than 16-36, 1-20, 95-103, 52-65, 81-100, and 103-110. Ability of the mabs to inhibit binding of [125I]hFSH to bovine testis membrane receptor (Rec) was also studied. When [125I]hFSH was preincubated with increments of each mab for 2 h at 25 C before adding Rec with further incubation for 16 h, all mabs inhibited [125I]hFSH binding to Rec. The data suggest that most of the hFSH beta molecule has a conformation enabling all antibody recognizable regions to be in close proximity to each other. The present study provides evidence for an assembled epitope comprising in part, amino acids 33-53, which has been previously shown to be involved in receptor binding. Peptide sequences 49-67 and 66-85 are neighboring sequences in this assembled epitope which contains the determinants for receptor binding.