We have previously described a cell-free assay that can be employed to study rejoining of radiation-induced DNA double-stranded breaks (dsb) in 'naked' DNA prepared from agarose-embedded cells using an extract of HeLa cells as a source of enzymes. Rejoining of dsb in this assay is absolutely dependent on cell extract and proceeds, under optimal reaction conditions, to an extent and with kinetics similar to those observed in intact cells. Here, we extend these experiments and demonstrate that the assay also supports rejoining of bleomycin and restriction endonuclease-induced dsb, agents that generate dsb with known ends. Rejoining of bleomycin-induced dsb proceeds to an extent and with kinetics similar to those observed with radiation-induced dsb. The kinetics of rejoining of restriction endonuclease-induced dsb are also similar to those of radiation-induced dsb. However, more and more dsb remain unrejoined as the extent of DNA fragmentation increases when enzymes cutting the DNA at frequent intervals are used. Dsb with blunt ends are rejoined with a similar efficiency to dsb with cohesive ends. Rejoining of restriction endonuclease-induced dsb is, in the presence of cell extract, more efficient than in the presence of T4 DNA ligase, suggesting the action in the overall reaction of activities in addition to DNA ligases. The experiments presented generalize the utility of the assay in studying the enzymology of dsb rejoining after treatment with radiomimetic drugs and restriction endonucleases and should be useful in the elucidation of the enzymatic requirements of dsb repair.