The MPM2 monoclonal antibody binds to a phospho amino acid-containing epitope present on more than 40 proteins of M-phase eukaryotic cells. We have developed a technique for cloning cDNAs encoding MPM2-reactive phosphoproteins from bacteriophage lambda expression libraries. Proteins from phage plaques were absorbed to nitrocellulose filters, phosphorylated by M-phase kinases, and screened for MPM2 binding. Partial-length cDNAs encoding two MPM2-reactive proteins termed MPM2-reactive phosphoproteins 1 and 2 (MPP1 and MPP2) were isolated. The deduced MPP1 and MPP2 amino acid sequences are not closely related to any previously described proteins. To determine which amino acid stretches contained the MPM2 epitope, sequences from a 15 amino acid peptide expression library were selected for binding to MPM2 after phosphorylation by M-phase kinases. A string of five amino acids was similar among all selected peptides, and the sequence reflecting the most frequent amino acid at each position was Leu-Thr-Pro-Leu-Lys (LTPLK). MPP1 and MPP2 proteins, respectively, contained five and nine sites closely related to LTPLK, including two that were common to both proteins, (F/T)TPLQ and SSP(I/S)D. Peptides containing LTPLK and FTPLQ were strongly phosphorylated by M-phase, but not interphase, cytosolic kinases, and the phosphorylated peptides were bound by MPM2. Thus, we have identified M-phase-specific phosphorylation sites bound by MPM2 and two putative M-phase phosphoproteins containing these sites.