Protein tyrosine kinase Yes is a cellular homolog of v-Yes, the oncogenic protein product of avian sarcoma virus Y73. Yes is a member of the Src family and its activation has been associated with several types of human cancer. Human Yes has not been previously characterized enzymatically. To carry out biochemical characterizations of this enzyme, we expressed it as a fusion protein with glutathione S-transferase in Escherichia coli, to allow purification in a single step. The affinity-purified GST-Yes has a specific activity of 1.3 nmol min-1 mg-1 with polyE4Y as substrate and Km values of 100 microg ml-1 for polyE4Y and 70 microM for ATP-Mg. The enzyme has a preference for magnesium over manganese ion for maximal activity. The divalent metal cation serves two essential functions for the activity of Yes: one as a part of the phosphate-donating substrate ATP-Mg and the other as an essential activator. The enzyme undergoes autophosphorylation without apparent activation. Finally, we show that the enzyme is inactivated by incubation with protein tyrosine kinase Csk in an ATP-Mg-dependent manner, indicating that cellular Yes can be regulated by Csk phosphorylation. These represent the first biochemical characterization of human Yes protein tyrosine kinase.