CP1 is a sequence-specific DNA-binding protein of the yeast Saccharomyces cerevisiae which recognizes the highly conserved DNA element I (CDEI) of yeast centromeres. We cloned and sequenced the gene encoding CP1. The gene codes for a protein of molecular weight 39,400. When expressed in Escherichia coli, the CP1 gene directed the synthesis of a CDEI-binding protein having the same gel mobility as purified yeast CP1. We have given the CP1 gene the genetic designation CEP1 (centromere protein 1). CEP1 was mapped and found to reside on chromosome X, 2.0 centimorgans from SUP4. Strains were constructed in which most of CEP1 was deleted. Such strains lacked detectable CP1 activity and were viable; however, CEP1 gene disruption resulted in a 35% increase in cell doubling time and a ninefold increase in the rate of mitotic chromosome loss. An unexpected consequence of CP1 gene disruption was methionine auxotrophy genetically linked to cep1. This result and the recent finding that CDEI sites in the MET25 promoter are required to activate transcription (D. Thomas, H. Cherest, and Y. Surdin-Kerjan, J. Mol. Biol. 9:3292-3298, 1989) suggest that CP1 is both a kinetochore protein and a transcription factor.