Five monoclonal antibodies to human (h) FSH have been prepared, isolated, and characterized. They were produced by hybridomas derived from FO myeloma cells and spleen cells from mice immunized with hFSH or its beta-subunit (hFSH beta). Two of the antibodies (A101 and A102) which recognized the alpha-subunit of hFSH bound much better when alpha was associated with the beta-subunit forming the intact hFSH molecule than when alpha was in free form. These antibodies showed 9-10%, 2-3%, and 1-3% cross-reactions with alpha-subunits in hTSH, hCG, and hLH, respectively. Two antibodies (B201 and B202) recognized only free beta-subunit. One antibody (B305) recognized free beta-subunit and native hFSH. There was no significant cross-reaction of these antibodies to FSH beta with hTSH, hLH, and hCG. Using solid phase competitive binding and sandwich assays, we compared the epitopes for these antibodies. Antibodies A101 and A102 recognize the same epitope on hFSH alpha. Antibodies B201 and B202 recognize different epitopes, but they seemed to be adjacent. Antibody B305 bound a different epitope than B201 and B202. Such characteristics of these antibodies can be useful for sensitive and specific assay of hFSH or hFSH beta and also may be helpful in studying FSH interaction with its receptor.