The nuclear matrix is increasingly identified with the processing of DNA damage. Previous work has suggested that association of DNA with the matrix can influence the repair of DNA double-strand breaks (DSBs) and the sensitivity of mammalian cells to ionizing radiation. By selectively examining DSBs that occur as multiples (multiple DSBs) within looped DNA structures, we have identified a subset of DSBs that repair with slow kinetics through the V(D)J recombination-associated DSB repair pathway. Enrichment of S-phase populations by centrifugal elutriation and selective examination of nascent DNA by pulse-labeling were used to demonstrate that elution of DNA from nucleoids is retarded by the presence of replicating DNA. Previously, application of a Poisson-based model of induction of multiple DSBs and DNA elution to a panel of mammalian cell lines indicated that the size of the looped chromatin domains varied between cell lines. The data presented here explain the range in domain sizes between cells as the result of differences in the percentage of cells actively replicating their DNA. Correction of the model to account for S-phase populations results in a looped domain size of 2.9 Mbp independent of cell type. Single-cell gel electrophoresis of nucleoids provides additional evidence for such sized structures. Stabilization of DNA to elution during S phase does not permit repair of DSBs in the DSB repair mutants xrs5 and St.SCID, both defective for the DSB repair pathway associated with V(D)J recombination.