SET, the translocation breakpoint-encoded protein in acute undifferentiated leukemia (AUL), is identified as a 39-kDa phosphoprotein found predominantly in the cell nuclei [1994, J. Biol. Chem. 269, 2258-2262]. SET is fused to a putative oncoprotein, CAN, in AUL and is thought to regulate the transformation potential of SET-CAN by its nuclear localization and phosphorylation. We investigated in detail the in vivo phosphorylation of SET. Phosphorylation of SET occurred in all human cell lines examined in vivo, primarily on serine residues. Endoproteinase Glu-C digestion of phosphorylated SET yielded two phosphopeptides. By radiosequencing, we identified the in vivo phosphorylation sites of SET as Ser9 and Ser24. The surrounding sequences of Ser9 and Ser24 contained an apparent consensus site sequence for protein kinase C.