The RFC4 gene encoding the 37-kDa subunit of Saccharomyces cerevisiae replication factor C (RF-C) has been cloned and sequenced. The RFC4 gene encodes a 323-amino acid polypeptide with a molecular weight of 36,126. The deduced amino acid sequence of the RFC4 gene shows high sequence similarity to the other two small subunits of yeast RF-C and the three small subunits of human RF-C (35-60% identity, 55-78% similarity). The similarity is greatest with the 40-kDa subunit of human RF-C (also called Activator 1). Despite the presence of a MluI cell cycle box in the regulatory region of the RFC4 gene, the steady state levels of its mRNA do not change significantly during the yeast mitotic cell cycle. The RFC4 gene is essential for yeast viability and is located on the left arm of chromosome XV, between the SUF1 and ADH1 genes. The essential role of this as well as the other small subunits of RF-C indicates a unique function for each of these subunits, despite their apparent structural redundancy. Rfc4p was overexpressed in Escherichia coli. Despite the presence of consensus ATPase motifs in the primary amino acid sequence, no discernible biochemical function could be ascribed to the purified protein. However, Rfc4p formed a tight complex with the product of the RFC3 gene which encodes the ATPase of RF-C.