A single mutation in the nucleotide binding pocket of select protein kinases allows for use of a bulky, substituted-ATP analog not used by the wild-type kinase [1]. Using this approach with the protein tyrosine kinase c-Src, we have generated a mutant T338G and expressed it in Src/Yes/Fyn null fibroblasts (SYF1) at near endogenous levels. T338G Sue exhibits high specificity for a substituted ATP analog N-6-2-phenyl ethyl ATP (peATP), which is not used by wild-type c-Src in autophosphorylation nor substrate phosphorylation assays. By employing the T338G Src mutant and [gamma-P-32]peATP analog, we demonstrate that c-Src can directly phosphorylate focal adhesion kinase (Fak) in vitro. We also show that incubation of permeabilized, T338 Src-expressing cells with pcATP causes an increase in Fak tyrosine phosphorylation not observed in wild-type Sue cells. Taken together, these data provide evidence that Src directly phosphorylates Fak and demonstrates the limitations of using this modified ATP strategy for analysis of direct substrates of protein kinases in permeabilized cells. (C) 2002 Elsevier Science (USA). All rights reserved.