Physical and functional interactions of human DNA polymerase α primase and replication protein A DNA polymerase alpha primase (pol prim) is a four subunit complex (p180 p68 p58 and p48). Pol prim is the only enzyme capable to start DNA synthesis de novo. It is evolutionary conserved from yeast to human and each subunit is essential for cell viability. The major role of pol prim is the initiation of DNA replication at chromosomal origins and in the discontinuous synthesis of Okazaki fragments on the lagging strand of the replication fork. There is a sequence similarity between the p58 primase subunit and the 8 kDa ssDNA binding domain of Pol ß. To determine whether p58 has any similar function a proteolytically stable domain of human primase CTp58 (G266 S510) containing the Pol ß like region was produced. It was shown that primase (p48 p58) and its components p48 and CTp58 bind to M13 ssDNA. Furthermore it was revealed that the CTp58 binds to oligonucleotides having different length and secondary structures. It was found that CTp58 binding not only depends upon the length but also on the structure of the oligonucleotide. This differential binding behavior with different structured oligonucleotides was further confirmed by primase assays. Moreover we showed that RPA and primase co operate in their binding to DNA. It was further found that iron is required for primase binding to DNA. Iron requirement was confirmed by a functional assay using primase devoid of iron and iron containing primase. These studies of a newly discovered DNA binding activity of DNA polymerase alpha primase led us to ask the question about the distribution and regulation of the enzyme complex in human cells. Therefore GFP tagged DNA polymerase alpha subunits p180 and p68 were analyzed in living as well as fixed cells. In both cases proteins were localized in the nucleus. Moreover RPA was also found in a complex with DNA polymerase alpha primase supporting the physical and functional interactions of DNA polymerase a primase and primase.