Prp20, a homolog to the mammalian negative regulator of chromosome condensation, RCC1, is retained on double-stranded (ds) DNA-cellulose when extracts are prepared from asynchronously growing wild-type yeast strains. Conversely, neither Prp20 from ts mutant cell extracts nor wt yeast Prp20 produced in Escherichia coli, bind to dsDNA-cellulose. In vitro reconstitution assays using E. coli-expressed Prp20 and inactivated ts mutant extracts of prp20-1 reveal that the Prp20 protein requires the assistance of other proteins in the cell extract to promote its binding to dsDNA. Immunoprecipitations and sizing-column-chromatography indicate that the Prp20 protein binds to the dsDNA column through a multicomponent complex composed of six to seven proteins, which has a collective molecular mass greater than 150,000 Da. At least three of the members of this Prp20 complex will bind GTP in vitro. Moreover, the Prp20 complex is shown to specifically lose its ability to bind dsDNA during the DNA replication phase of the cell cycle. This loss of dsDNA binding during the S phase of the cell cycle does not affect the proper organization of the nucleoplasm and appears to be reversed before the cell enters mitosis.