The actin-filament-associated protein (AFAP-1 10) forms a stable complex with activated variants of the Pp60c-src (Src) non-receptor tyrosine kinase through SH2 and SH3 interactions. In this report, site-directed mutagenesis and a transient expression system that permits co-expression of activated pp60c-src (Src527F) and AFAP-110 in Cos-1 cells were used to identify the SH2-binding motif in AFAP-110. Four tyrosine residues, two in the amino terminus (Y93 and Y94) and two in the carboxy terminus (Y451 and Y453), were mutated to phenylalanine, significantly reducing overall steady-state levels of tyrosine phosphorylation and preventing Src527F from forming a stable complex with AFAP-110. These data indicate that the major sites for tyrosine phosphorylation are among these four tyrosine residues and that one or more of these tyrosines may function as an SH2-binding motif. Mutagenesis of just two tyrosines in either the amino terminus (Y93/Y94) or in the carboxy terminus (Y451/Y453) to phenylalanine had only a modest effect on steady-state levels of tyrosine phosphorylation and was not sufficient to abrogate stable-complex formation. These data suggest that Src527F can form a stable complex with AFAP-110 through either of two independently functional SH2-binding motifs. Triple-tyrosine mutation demonstrated that Y93 was not significantly phosphorylated on tyrosine and would not facilitate stable complex formation, whereas Y94, Y451, and Y453 could be phosphorylated on tyrosine and would facilitate stable-complex formation. We hypothesize that Src527F and AFAP-110 interact through a multistep binding mechanism that may either extend interactions between Src527F and actin filaments or permit reorientation of Src527F on AFAP-110, which could facilitate the presentation of Src527F toward other signaling molecules.