In the nuclei of eukaryotic cells, initiation of DNA replication occurs at a discrete number of foci. One component of these foci is the DNA replication factor RP-A. Here, the process leading to the association of RP-A with foci was reconstituted with cytosolic fractions derived from Xenopus eggs. With the use of this fractionated system, a 170-kilodalton protein required for the assembly of RP-A into foci was identified and purified. The protein appears to be an integral component of the foci at which replication of DNA is initiated in eukaryotic nuclei.