Csk (C-terminal Src kinase) is a protein tyrosine kinase that phosphorylates Src family member C-terminal tails, resulting in down-regulation of Src family members. The molecular basis of Csk's substrate specificity and catalytic mechanism with a protein substrate was investigated. Using a peptide library approach, preferential amino acids which are unrelated to the conserved Src C-terminal sequence were identified. The validity of these preferences was confirmed by synthesizing a short consensus peptide and demonstrating its high catalytic efficiency with Csk. These results underscore the difficulties of relying on amino acids neighboring tyrosine in protein sequences as predictors of protein kinase substrate specificity for in vivo analysis. In addition, a catalytically inactive version of the Src family member, Lck (lymphoid cell kinase), was expressed, purified, and evaluated as a Csk substrate. It was proven to be the most catalytically efficient substrate yet identified for Csk. The high efficiency of purified Csk phosphorylating a pure, unphosphorylated Src family member argues against the importance of an SH2-phosphotyrosine docking interaction or the involvement of extra recruitment proteins in facilitating Csk phosphorylation of Src family members. Kinetic studies revealed that the chemical step is at least partially rate-determining in Csk-mediated phosphoryl transfer to the Lck protein. Other properties including preferences for Mn over Mg, thio effects, and Km's for ATP also correlate fairly well between protein and peptide phosphorylation. The lack of a significant impact of increased salt on the Km for Lck phosphorylation differs from Csk-mediated poly(Glu,Tyr) phosphorylation, and argues against the importance of electrostatic effects in the Csk-Lck binding interaction. The failure of the Lck phosphorylation product (phosphotyrosine-505) to significantly inhibit Csk phosphorylation of Lck is consistent with a catalytic model involving multidomain structural interactions between substrate and enzyme.