The formation of triple-helical DNA has been implicated in several cellular processes, including transcription, replication and recombination. While there is no direct evidence for triplexes in vivo, cellular proteins that specifically recognize triplex DNA have been described. Using a purine-motif triplex probe and southwestern library screening, we isolated five independent clones expressing the same C-terminal 210 amino acids of the Saccharomyces cerevisiae protein Cdp1p fused with beta-galactosidase. In electrophoretic mobility shift assays, recombinant Cdp1pDelta1-867 bound Pu-motif triplex DNAs with high affinity (K:(d) approximately 5 nM) and bound Py-motif triplex, duplex and single-stranded DNAs with far lower affinity (0.5-5.0 microM). Genetic analyses revealed that the CDP1 gene product was required for proper chromosome segregation. The possible involvement of triplex DNA in this process is discussed.