Hsp90 is gaining increasing importance as a protein involved in controlling the normal functioning of the cell. To do this it apparently interacts with a battery of co-chaperone proteins that are involved in both substrate recognition and the progression of the Hsp90 catalytic pathway. In this report we have identified the Drosophila Dpit47 protein (DNA polymerase interacting tpr containing protein of 47 kDa) through its interaction with the DNA polymerase alpha. This protein is a predominantly nuclear protein, which forms a tight and stoichiometric interaction with Hsp90 and shows interaction with Hsp70. It also has substantial homology to other known Hsp90 co-chaperones, e.g. CNS1 and hop1, making it likely that this protein also functions as an Hsp90 co-chaperone. The interaction with the DNA polymerase alpha is not related to the special situation in early embryos where there are large amounts of maternal protein stockpiles of the polymerase, as it occurs to the same level in early and late embryos and also in proliferating cell culture. However, it does not occur in quiescent cells, making it likely that the protein is related to proliferation. This is also consistent with Dpit47 expression being higher in proliferating cells. The interaction between the Dpit47 and the polymerase takes place predominantly in the nucleoplasm, and seems to involve several subunits of the polymerase in comparable amounts, making it unlikely that it is solely required for the assembly of the polymerase complex. The polymerase can also be seen to interact with Hsp90, and the interaction between Dpit47 and the polymerase is increased by the specific Hsp90 inhibitor geldanamycin. This suggests that a complex of the Dpit47, Hsp90 and DNA polymerase exists in the cell. The interaction between DNA polymerase alpha and Dpit47 completely inhibits the activity of the polymerase. These results suggest that Hsp90 acts as a chaperone for DNA polymerase alpha and that this interaction is mediated through the novel co-chaperone Dpit47. This provides the first suggestion of a role for chaperones in DNA replication in higher eukaryotes.