The actin filament-associated protein, AFAP-110, is a Src SH2/SH3 binding partner that can modulate changes in actin filament structure. AFAP-110 contains a carboxy terminal motif that facilitates actin filament interactions, as well as amino terminal protein binding motifs, including an SH3 binding motif, two SH2 binding motifs, and two Pleckstrin homology domains. Two monoclonal antibodies (MAbs) were developed that recognized epitopes in either the amino terminus (MAb 4C3) or the carboxy terminus (anti-AFAP-110) of AFAP-110. Site-directed mutations that change key proline residues to alanine in the SH3 binding motif and an adjacent proline-rich motif abrogated MAb 4C3 binding. These same mutations have been shown to prevent SH3 interactions between AFAP-110 and Src527F. These data indicate that MAb 4C3 recognizes an epitope that is part of the SH3 binding motif. Interestingly, MAb 4C3 is not efficiently reactive with mammalian homologs of AFAP-110. Sequence analysis of a putative cDNA clone that encodes the amino terminus of the human AFAP-110 isoform predicted a one amino acid difference within this epitope, indicating a mechanism for species-specific binding by MAb 4C3. A second, MAb anti-AFAP-110, recognizes AFAP-110 across species and binds to an epitope within the carboxy terminus. This epitope includes the 5th heptad repeat of the carboxy terminal, leucine zipper motif (amino acids 592-598)--a motif that facilitates self-associations and may regulate the function of AFAP-110. These MAbs will be useful for analyzing the effects of AFAP-110 upon cell morphology and actin filament integrity. In addition, the avian-specific MAb 4C3 may be useful for studying the effects of avian AFAP-110 constructs expressed in mammalian cells, by providing an internal epitope tag.