||Src family kinases are non-receptor protein tyrosine kinases that are localized at the periphery of the cellular membrane, via their N-terminal structures. They are expressed at high levels in various tissues and play important roles in receptor- mediated signal transduction. Oncogenic forms of various members of this family have been detected. We show that rat brain contains a factor that strongly inhibits the activity of Src family protein kinases including bacteria expressed GST-Lck, GST-Fyn and Fyn from bovine thymus tissue. This factor does not recognize protein tyrosine kinase GST-Wee1 and serine/threonine kinases Mst2, Cdk5/p25 or Cdk5/p35, Cdk2/cyclinA. The inhibitory activity is heat labile and dialyzable. A series of tests have been conducted, ruling out the possibility that the inhibition was a direct result of the presence of ATPase, phosphatase or protease. Mutant GST-Lck with normal C-terminal tyrosine substituted with phenyalanine is also inhibited by this factor to an extent similar to that of wild type GST-Lck. Addition of ATP/Mg2+ to the pre-incubation of GST-Lck and inhibitory factor does not increase the inhibition rate but decrease it. These results indicate the inhibitory factor is not Csk and is able to inhibit oncogenic form of Src family kinase. Gel filtration chromatography indicates that the molecular size of this unknown inhibitor in rat brain is around 44 kD. Elution of GSH-beads bound with inactivated GST-Lck elutes the decreased activity, indicating an irreversible inhibition between the inhibitor and GST-Lck. The inhibition of Fyn from bovine thymus by this inhibitor is reversible. Rat thymus contains a similar inhibitory factor but with about 20-fold higher specific activity. An inhibitory activity with potency similar to that of rat thymus exists in bovine spleen. For purification purpose, the sequential combination of DEAE Fast Flow chromatography, Hydroxyapatite chromatography and Sephadex 200 Gel filtration chromatography results in an approximately 188-fold purification of the inhibitor in bovine spleen. 5.0 ng of the peak fraction from Sephadex 200 makes 50% inhibition on GST-Lck activity.