In vivo, nucleosomes are formed rapidly on newly synthesized DNA after polymerase passage. Previously, a protein complex from human cells, termed chromatin assembly factor-I (CAF-I), was isolated that assembles nucleosomes preferentially onto SV40 DNA templates that undergo replication in vitro. Using a similar assay, we now report the purification of CAF-I from the budding yeast Saccharomyces cerevisiae. Amino acid sequence data from purified yeast CAF-I led to identification of the genes encoding each subunit in the yeast genome data base. The CAC1 and CAC2 (chromatin assembly complex) genes encode proteins similar to the p150 and p60 subunits of human CAF-I, respectively. The gene encoding the p50 subunit of yeast CAF-I (CAC3) is similar to the human p48 CAF-I subunit and was identified previously as MSI1, a member of a highly conserved subfamily of WD repeat proteins implicated in histone function in several organisms. Thus, CAF-I has been conserved functionally and structurally from yeast to human cells. Genes encoding the CAF-I subunits (collectively referred to as CAC genes) are not essential for cell viability. However, deletion of any CAC gene causes an increase in sensitivity to ultraviolet radiation, without significantly increasing sensitivity to gamma rays. This is consistent with previous biochemical data demonstrating the ability of CAF-I to assemble nucleosomes on templates undergoing nucleotide excision repair. Deletion of CAC genes also strongly reduces silencing of genes adjacent to telomeric DNA; the CAC1 gene is identical to RLF2 (Rap1p localization factor-2), a gene required for the normal distribution of the telomere-binding Rap1p protein within the nucleus. Together, these data suggest that CAF-I plays a role in generating chromatin structures in vivo.