Yeast replication factor C (RF-C) is a muttipolypeptide complex required for chromosomal DNA replication. Previously this complex was known to consist of at least four subunits. We here report the Identification of a fifth RF-C subunit from Saccharomyces cerevisiae, encoded by the RFC5 (YBR0810) gene. This subunit exhibits highest homology to the 38 kDa subunit (38%) of human RF-C (activator 1). Like the other four RFC genes, the RFC5 gene Is essential for yeast viability, indicating an essential function for each subunit. RFC5 mRNA is expressed at steady-state levels throughout the mitotic cell cycle. Upon overexpression in Escherichla coli Rfc5p has an apparent molecular mass of 41 kDa. Overproduction of RF-C activity in yeast is dependent on overexpression of the RFC5 gene together with overexpression of the RFC1-4 genes, indicating that the RFC5 gene product forms an integral subunit of this replication factor.