Replication protein A (RPA) is a three-subunit protein that plays a central role in eukaryotic DNA replication, recombination, and repair. We have previously reported the cloning and bacterial expression of the 70- and 32-kDa subunits of human RPA (hRPA). We have now cloned the 14-kDa subunit (hRPA3) from a HeLa cell cDNA library. The hRPA3 cDNA is a 692-base pair sequence that contains an open reading frame encoding a protein of 121 amino acids with a calculated molecular mass of 13.6 kDa. The deduced amino acid sequence shows only limited similarity to the small subunit of yeast RPA and is unrelated to any other protein in the current data banks. A recombinant protein containing a short histidine tag at the NH2 terminus has been purified in good yield from Escherichia coli by metal-chelate affinity chromatography. Antibodies prepared against recombinant hRPA3 recognize the native protein and inhibit SV40 DNA replication in vitro. We have localized the genes for the 70-, 32-, and 14-kDa subunits to chromosomes 17, 1, and 7, respectively, using polymerase chain reaction amplification of genomic DNA from rodent-human hybrid cell lines. Since RPA appears to be involved in several fundamental cellular processes, the physical mapping of the RPA genes may be useful in identifying possible human genetic defects associated with RPA deficiency or dysfunction.