C-Yes is a non-receptor-type tyrosine kinase of the Src family that is most closely related to c-Src, C-Yes has been implicated in development of some human cancers. Here we report on the expression, purification, and characterization of the active human recombinant c-Yes. A full-length human c-Yes clone has been. generated and the protein was expressed in insect Sf9 cells. Active c-Yes was purified by liquid chromatography to yield a preparation with a high specific activity (160 nmol/min/mg using an optimal Src substrate peptide). In a comparison between human c-Yes and c-Src enzymes, relative phosphorylation efficiencies on nine protein and four peptide substrates were different. However, the recently described Src inhibitor CGP77675 inhibited human c-Yes with a potency similar to that of c-Src (IC50 value of 6.5 nM). The purified preparation of active c-Yes provides a good basis for further enzymatic characterization and for the development of c-Yes-specific inhibitors,