Bcr is a novel serine/threonine protein kinase that is believed to require two cysteine pairs for activity (Maru and Witte, Cell, 67, 459, 1991). Tyrosine phosphorylated Bcr has dramatically reduced kinase activity, and tyrosine 360 of Bcr, which is one of the sites of phosphorylation by the Bcr-Abl oncoprotein, is required for transkinase activity (Liu et al., Mol. Cell Biol., 16, 998, 1996). Results presented here indicate that Bcr tyrosine 328 is also phosphorylated within Bcr-Abl expressing cells and is required for Bcr's serine/threonine kinase activity. Bcr Y328F, like Bcr Y360F, had defective transkinase activity but can autophosphorylate. However, the Y328F/Y360F double mutant of Bcr is defective in both trans- and autokinase activities. Taken together with the kinase inhibitory effects of tyrosine phosphorylation of Bcr by Bcr-Abl, our studies with tyrosine to phenylalanine Bcr mutants indicate that the hydroxyl residues of tyrosines 328 and 360 play crucial roles in Bcr's kinase activity.