We have previously shown that stimulation of 32D cl3 cells with interleukin (IL)-3 results in the activation of three src-like tyrosine kinases, fyn, hck, and lyn. The subunit of the IL-3 receptor co- immunoprecipitated with hck in lysates of both unstimulated and IL-3- stimulated cells; however, the β subunit did not precipitate with either fyn or lyn. The association of these three kinases with the β subunit of the IL- 3 receptor was further investigated using bacterial fusion proteins encoding the unique, SH3, and SH2 domains of these three kinases. Fusion proteins of both hck and fyn bound to a 150-kDa tyrosine-phosphorylated protein present in lysates of IL-3-stimulated cells. This protein was identified as the β subunit of the IL-3 receptor by immunoblotting with an anti-β antibody. Glutathione S-transferase (GST) fusion proteins containing the SH2 domain of hck bound to the β subunit although the amount of β subunit that bound to the SH2 domain alone was only 30% of that which bound to the fusion protein containing the unique, SH3, and SH2 domains. This indicates that the SH2 domain is one of the motifs involved in binding hck to the β subunit. A GST fusion protein encoding a 236-amino acid region of the cytoplasmic tail of the β sub-unit, which contained four tyrosine residues, bound to hck and fyn. Binding to both proteins was dramatically increased when the GST-β fusion protein was tyrosine-phosphorylated. Far Western blot analysis was used to demonstrate the binding of the unique, SH3, and SH2 domains of hck to this 236-amino acid region of the subunit; tyrosine phosphorylation of this protein increased the binding of both the unique region and the SH2 domain probes. These data indicate that binding of hck to the β subunit is mediated by both phosphotyrosine-dependent and -independent mechanisms.