CrkII is an intracellular adaptor protein involved in signal transduction by various growth factors. Activation of PDGF α-receptor resulted in its association with CrkII in vivo. In contrast, binding of CrkII to the PDGF β-receptor was negligible, despite its becoming prominently phosphorylated. Bacterially expressed GST-CrkII SH2 domain specifically bound to Tyr-762 and Tyr-771 in the activated PDGF α- and β- receptors, respectively. GST fusion protein of full-length CrkII also bound to the activated PDGF β-receptor. However, tyrosine phosphorylation of GST-CrkII diminished its binding to the β-receptor. CrkI, a truncated version of CrkII lacking the phosphorylatable tyrosine residue, could bind to both PDGF α- and β-receptors in vivo. In conclusion, tyrosine phosphorylation of CrkII negatively affects its binding to the PDGF receptors. The differential binding of CrkII to the PDGF α- and β- receptors may be a rationale for functional diversity between the two receptors.