The c-src and c-yes proto-oncogenes encode 60 000 and 62 000 Dalton non-receptor tyrosine kinases of the Src family, pp60c-src and pp62c-yes, respectively. These kinases are over 80% homologous outside of their unique amino termini, yet several studies suggest that differences exist in the regulation, activation, and function of cSrc and cYes. The determinants of specificity in signaling between these proteins, however, remain unclear. In order to investigate the roles of the Src Homology (SH) 3 and 2 domains in mediating signaling specificity between cSrc and cYes, chimeras were created in which the SH3 and/or SH2 domains of cSrc or the fully activated variant Src527F were replaced by the corresponding domains of cYes. These constructs were used to assess the effects of the Yes SH3 and SH2 domains on the ability of Src to form stable complexes with and induce tyrosine phosphorylation of Src SH3 and SH2 domain binding partners in vivo. Both the Yes SH3 and SH2 domains were found to alter the capacity of Src to form stable associations with heterologous proteins. The Yes SH3 domain was unable to affinity absorb the Src SH3/SH2 binding partner AFAP-110 from COS-1 cell lysates, and chimeric constructs of Src527F containing the cYes SH3 domain were unable to efficiently co-immunoprecipitate with AFAP-110 from chicken embryo fibroblasts. Interactions with the Src SH2 domain binding partner pp130cas were unaffected. Additionally, only chimeras containing the cYes SH2 domain were able to co-immunoprecipitate with an unidentified 87 kDa tyrosine-phosphorylated protein. These results indicate that the SH3 and SH2 domains are capable of directing specificity in substrate binding between Src and Yes, suggesting potential mechanisms for generating specificity in signaling between these two highly related non-receptor tyrosine kinases.