The members of the Src kinase family are expressed in a wide variety of tissues, but some of them such as Blk, Hck, Fgr, Lck and Lyn are found primarily in hematopoietic cells. In the present study, we have undertaken experiments to test whether Src kinase cleavage and relocation is a general mechanism during induction of apoptosis. Our results indicate that Fyn and Lyn are efficiently cleaved in their unique region in hematopoietic cells undergoing apoptosis. Fyn cleavage occurred in Fas-stimulated Jurkat T cells but Fyn and Lyn were also processed in the SKW6.4 B cell line. Inhibition of caspases by Z-VAD-fmk or Ac-DEVD-CHO totally prevented Fyn and Lyn cleavage in both intact cells and in vitro. Fyn and Lyn but not Lck, Src and Hck were processed in vitro by human recombinant caspase 3 and by cellular extracts prepared from Fas-stimulated cells. Single mutation of Asp 19 or Asp 18 in the unique N-terminal domains of Fyn and Lyn respectively abolished their cleavage and relocation into the cytoplasm of apoptotic cells. When immunoprecipitated from COS cells N-terminal deleted Src kinases exhibited increased enzymatic kinase activity toward enolase. Thus, cleavage of Fyn and Lyn during induction of apoptosis represents a new mechanism for the regulation of Src kinases that may have important functional and physiological consequences.