The product of the Saccharomyces cerevisiae MCK1 gene is a protein kinase that phosphorylates poly (Glu,Tyr) in vitro and is itself phosphorylated at both tyrosine and serine in vivo. To characterize the substrate specificity of Mck1, the enzyme was purified to apparent homogeneity from the soluble fraction of yeast cell extracts by ammonium sulfate precipitation, followed by ion exchange chromatography (Q- and S-Sepharose), dye-ligand affinity chromatography (Orange A-agarose), adsorption chromatography (hydroxylapatite), and ion exchange fast protein liquid chromatography (Mono-S). In the absence of an exogenous substrate, purified Mck1 was able to autophosphorylate on tyrosine and serine. A catalytically inactive mutant (K68R in conserved kinase domain II) expressed in an mck1 delta strain did not contain detectable phosphotyrosine, confirming that the tyrosine phosphorylation observed in vivo is due to autophosphorylation, but did contain phosphoserine, suggesting that Mck1 is a target for other cellular protein kinases. Purified Mck1 phosphorylated a variety of proteins in heat-inactivated yeast extracts, primarily on serine (and threonine). The purified enzyme also used a number of mammalian proteins as phosphoacceptors, including myelin basic protein (MBP), microtubule-associated protein 2 (MAP-2), and tau protein. All of these substrates were phosphorylated on either serine or threonine (or both). Mck1 isolated from yeast extracts by immunoprecipitation with an anti-Mck1 antibody directed against its C terminus also phosphorylated MBP at serine. In the same immune complex kinase assay, the K68R mutant did not detectably phosphorylate MBP, indicating that the serine-specific phosphotransferase activity of Mck1 is intrinsic and not due to contamination by an associated kinase. These findings demonstrate that Mck1 is a member of a novel class of protein kinases that displays the ability to phosphorylate all three hydroxyamino acids in proteins.