Antibody-secreting hybridoma cell lines produced from BALB/c mice that had been immunized with horse cytochrome c (cyt c) conjugated to hemocyanin yielded six hybridoma subclones that produced four monoclonal antibodies (mAb) with different patterns of cross-reactivity with a panel of evolutionarily variant cyt c. The recognition sites for three of these mAb lay in the same region of the intact molecule, because two of the mAb were sensitive to the amino acid residue present at sequence position 44, with one requiring threonine at position 47. The fourth mAb bound in another region of the molecule at a site that involves either residue 60 or residue 89. Synthetic peptides that included these residues did not react with these mAb, indicating that these sites may require interactions from noncontiguous regions of the molecule to bind antibody. The association constants for the interaction of the mAb with horse cyt c were very similar and of the order of 10(10) M-1. Specificity studies with anti-idiotypic sera and competition assays between mAb for binding to horse cyt c confirmed that the six positive hybridoma subclones produced from this fusion produced mAb that had one of these four distinct specificities. The idiotypes of these four mAb were serologically distinct, and were derived from Vh genes of the J558 family.