We described previously a cell-free assay, that could be employed to study the rejoining of radiation-induced DNA double-strand breaks (dsb) in agarose embedded nuclei by activities present in an extract prepared from exponentially growing HeLa cells. Here, we extend the study and present an in vitro assay for rejoining of radiation-induced DNA dsb that employs 'naked' DNA prepared from agarose-embedded cells as a substrate and extract of HeLa cells as an enzyme source. There is no detectable residual protein on substrate DNA after extensive lysis with ionic detergents and treatment with proteases, as determined by SDS-PAGE and silver staining. We demonstrate that rejoining of dsb is absolutely dependent on cell extract and that, under optimal reaction conditions, it proceeds to an extent and with kinetics similar to those observed in intact cells. Dsb rejoining in this assay requires Mg 2+ and is inhibited by high concentrations of either K+ or Na+. This assay complements the nuclei assay for DNA dsb repair previously developed, and may be preferable to the latter in the purification of factors involved in DNA dsb repair, as it employs as substrate DNA deprived of proteins.