We have studied the activity and substrate specificity of the catalytic domain of a protein kinase that was isolated in a screen of a human lambda gt11 fibroblast cDNA library with anti-phosphotyrosine antibodies. The sequence of this protein kinase would predict that it is a protein serine/threonine kinase, which at first seemed incongruent with the cloning method. However, recent reports indicate that some protein kinases can phosphorylate both tyrosine and serine/threonine residues. To determine whether this protein kinase, which we call PYT (for phosphotyrosine picked threonine kinase), was a dual-specificity protein kinase we investigated its substrate specificity when expressed in bacteria. The catalytic domain was active as a protein kinase when expressed from any of several promoters and when expressed as a TrpE fusion protein. All experiments that resulted in an active protein kinase, as judged by incorporation of 32P by metabolic labeling, also resulted in the generation of proteins that were recognized by anti-phosphotyrosine antibodies. Phosphoamino acid analyses of the metabolically labeled proteins that were recognized by the antibodies consistently yielded large amounts of phosphothreonine and only trace amounts of phosphotyrosine. We mapped the phosphorylation sites in the phosphorylated PYT protein and found only phosphothreonine; 90% of the radioactivity mapped to a threonine in the region autophosphorylated by many protein kinases. These data demonstrate that PYT is primarily a protein threonine kinase, but that it can phosphorylate tyrosine to a small extent, making it a potential dual-specificity protein kinase.