CP1 (encoded by CEP1) is a Saccharomyces cerevisiae chromatin protein that binds a DNA element conserved in centromeres and in the 5'-flanking DNA of methionine biosynthetic (MET) genes. Strains lacking CP1 are defective in chromosome segregation and MET gene transcription, leading to the hypothesis that CP1 plays a general role in assembling higher order chromatin structures at genomic sites where it is bound. A screen for mutations synthetically lethal with a cep1 null allele yielded five recessive csl (cep1 synthetic lethal) mutations, each defining a unique complementation group. Four of the five mutations synergistically increased the loss rate of marker chromosomes carrying a centromere lacking the CP1 binding site, suggesting that the cep1 synthetic lethality was due to chromosome segregation defects. Three of these four CSL genes were subsequently found to be known or imputed kinetochore genes: CEP3, NDC10, and CSE4. The fourth, CSL4, corresponded to ORF YNL232w on chromosome XIV, and was found to be essential. A human cDNA was identified that encoded a protein homologous to Csl4 and that complemented the csl4-1 mutation. The results are consistent with the view that the major cellular role of CP1 is to safeguard the biochemical integrity of the kinetochore.