The role of specific amino acid residues within the sequence region between Leu26 and Thr46 of the human follicle-stimulating hormone (hFSH) alpha-subunit in the FSH-receptor interaction has been investigated. Competitive binding activities of seven sets of synthetic peptides were evaluated with both FSH- and luteinizing hormone (LH)-radioreceptor assay procedures. Set 1 included two overlapping peptides (alpha 25-41 and alpha 31-45) spanning the alpha 26-46 region, while Sets 2-6 included peptides of different size or structure in which the alpha 26-46 sequence was (i) sequentially truncated either from the N-terminus or from the C-terminus or both; (ii) alternatively reduced to a series of overlapping 13-mer peptides; or (iii) modified at the C-terminal Arg and Lys residues with substitution by Ala residues. In Set 7, synthetic peptides related to the parent alpha 26-46 peptide were prepared in which all the cysteine residues were substituted either singly or multiply with serine residues (i.e., alpha 26-46 Cys28,31,32-->Ser28,31,32). The ED50 values of the parent alpha 26-46 peptide in the FSH-radioreceptor assay were 2 and 7 x 10(-5) M, depending on whether the C-terminus was present as the amide or the free acid form, respectively. The substituted peptide alpha 26-46 (Cys28,31,32-->Ser28,31,32) was totally inactive in the FSH-radioreceptor assay. The truncation studies indicated that Cys28, Cys31 and Cys32 all contribute to the hormone-receptor interaction, with Cys32 being the major contributory cysteine residue. Similar results were observed when these peptides were evaluated in the LH-radioreceptor assay where the ED50 value for the parent alpha 26-46 peptide was observed to be 2 or 3 x 10(-5) M, depending on whether the C-terminus was the amide or the free acid. The Cys31 residue did not appear to contribute to the LH-receptor interaction; however, removal of Cys28 and Cys32 resulted in significant decreases in binding activity. The C-terminal truncation studies of the alpha 26-46 peptide revealed that Lys44 contributes to FSH receptor binding activity but does not contribute to the LH-receptor interaction. Truncation of the Arg42 residue or substitution of Arg42 with alanine in the alpha 31-45 peptide sequence prepared as part of the Set 1 synthetic peptides (i.e., the alpha 31-45 Arg42-->Ala42 peptide) or as part of the Set 6 peptide series, alpha 26-46 Arg42-->Ala42, confirmed the involvement of this residue in the LH-receptor interaction.(ABSTRACT TRUNCATED AT 400 WORDS)