Three different experimental approaches were used to assess the regions on the beta-subunit of human FSH (hFSH beta) that may be altered or masked by its association with the alpha-subunit of hFSH (hFSH alpha) in the heterodimeric hFSH molecule. In a direct approach, we tested whether synthetic peptides corresponding to hFSH beta sequences 1-20, 16-36, 33-53, 49-67, 66-85, 81-100, and 98-111 inhibited association of hFSH alpha and hFSH beta in an enzyme-linked immunosorbent assay. Synthetic peptides-(81-100), -(98-111), and -(66-85) caused greater than 50% inhibition of subunit association, whereas other peptides showed 26% or less inhibition. These data suggested that the C-terminal sequences of hFSH beta, particularly 81-100, are at a subunit interface with hFSH alpha in heterodimeric hFSH. In another approach we reasoned that antibodies with a higher affinity for free hFSH beta than for heterodimeric hFSH bind to epitopes on hFSH beta that are masked or altered by hFSH alpha subunit. To test this hypothesis, epitopes of hFSH beta were mapped using synthetic peptides of hFSH beta sequences, three monoclonal antibodies (3G3, 4D5, and 4G8), and a polyclonal antiserum (NIDDK anti-hFSH beta). Compared to 3G3 all the other antibodies exhibited minimal reactivity with hFSH, but bound strongly to hFSH beta. The epitope-mapping data with both 4D5 and NIDDK anti-hFSH beta identified peptide 81-100, which was not recognized by 3G3. The epitope map with 4G8 identified the same three peptides as with 3G3. However, in the case of 4G8 its reactivity with peptide 33-53 was the least, whereas it was ranked first for 3G3. Since both 3G3 and 4G8 had an identical affinity for hFSH beta, it was hypothesized that sequences in peptide 33-53 may be altered or masked by hFSH alpha. To test this, we determined the specificity of anti-hFSH beta-(33-53) peptide antiserum for hFSH beta and hFSH in an enzyme-linked immunosorbent assay. The antipeptide antiserum bound strongly to free hFSH beta and weakly to hFSH, suggesting that part of the sequence in peptide-(33-53) was masked or altered by association with hFSH alpha in heterodimeric hFSH. Taken together, the subunit association studies, the epitope-mapping data, and the specificity of anti-hFSH beta-(33-53) peptide antiserum have suggested that sequences in peptide-(81-100) and -(33-53) are masked or conformationally altered by hFSH alpha in heterodimeric hFSH.