Structure-function investigations were undertaken to increase understanding of the surface topology of the alpha-subunit of human FSH (hFSH). The objectives were to determine which sequences of the alpha-subunit of hFSH are surface-oriented (exposed to antibody) and to identify which of these surface-oriented sequences are in contact with the beta-subunit of hFSH in the alpha/beta heterodimer. Seven overlapping synthetic peptides spanning the primary structure of hFSH alpha were used for immunizing rabbits to generate site-specific antipeptide antisera. The antisera were characterized with respect to their reactivity to the seven synthetic peptides, as well as hFSH, hFSH alpha, hFSH beta, and hFSH alpha r/a (reduced and alkylated), using an enzyme-linked immunosorbent assay. All of the peptides successfully generated antipeptide antisera with titers that range from 1:1,600 to 1:80,000. Anti-1-15 bound exclusively to hFSH alpha. Anti-11-27 and anti-33-58 bound to hFSH alpha to a much greater extent than to hFSH. In contrast, anti-73-92 had only slightly higher binding to hFSH alpha than to hFSH. Anti-22-39, anti-51-65, and anti-61-78 all failed to bind to either hFSH or hFSH alpha. With the exception of anti-22-39, all of the remaining antisera bound to hFSH alpha r/a. None of the antisera bound to hFSH beta. These data strongly suggest the following. Sequences 1-15, 11-27, and 33-58 contain residues that are masked by hFSH beta and are thus in or near the alpha/beta-subunit interface. In addition, sequences 11-27 and 33-58 contain other distinct residues that are surface-oriented in the hFSH heterodimer. In contrast, sequence 73-92 appears to be surface-oriented in the hFSH heterodimer. Lastly, sequences 51-65 and 61-78 appear to be buried within the native alpha-subunit and, thus, are unable to interact with antibodies. These results agree with and extend previous findings and will prove useful to those currently investigating the surface topology and structure-function relationships of the glycoprotein hormones.