The catalytic domain of chicken Src including the C-terminal tail (Src-CD), has been expressed in Schizosaccharomyces pombe and purified to homogeneity. The expressed protein is a mixture of unphosphorylated (80%) and mono-phosphorylated (20%) species, that can be separated from each other by Mono Q chromatography. By a novel mass spectrometric method that utilizes parent ion scans of unseparated peptide mixtures, we found that the mono-phosphorylated form is phosphorylated either at Tyr416 or at Tyr436. The stability of Src-CD is comparable to the wild-type protein. Src-CD auto-phosphorylates and efficiently phosphorylates substrate peptides and proteins. Auto-phosphorylation occurs by an intermolecular mechanism and is completely inhibited by an excess of substrate peptide. Kinetic measurements for two exogenous substrates, the Src substrate peptide (AEEEIYGEFEAKKKK) and denatured enolase, showed that the overall activity (kcat) of the Src-CD molecule is about 10 times higher than that of wild-type Src. The kcat values for phosphorylation of the Src substrate peptide are similar for the unphosphorylated and monophosphorylated Src-CD (50 min-1), but the apparent K(m) values differ significantly (approximately 3 microM and 10 microM, respectively). Therefore, at low substrate concentrations in vitro the mono-phosphorylated form is more active, in agreement with the importance of Tyr416 for in vivo activity. The apparent K(m) values of the mono-phosphorylated Src-CD and wild-type Src for the Src substrate peptide and enolase are similar, indicating that, under these conditions, the kinase domain is mainly responsible for substrate binding.