We have reported the existence in rat nuclear extracts of a specific cleavage activity on a DNA fragment containing the human minisatellite MsH42 region (minisatellite plus its flanking sequences). Here, we have developed a system to analyse the nature of the cleavage products from the MsH42 region generated by the nuclear extracts. Our results demonstrated the formation of DNA double-strand breaks (DSB) in the MsH42 region by two different enzymatic activities, and that their distribution along this fragment changes depending on the presence of Mg2+. In the assays with Mg2+, the DSB were located in the minisatellite and its 3'-flanking region, showing preference for G-rich stretches. Oligonucleotide mutagenesis analysis confirmed that this enzymatic activity has a strong preference for G-tracts and that the recognition site is polarized towards the 3' end. Moreover, this activity cuts GC palindromes efficiently. In contrast, in the experiments without Mg2+, most DSB were mapped within the minisatellite flanking sequences. The analysis with oligonucleotides showed that G-tracts are recognized by this endonuclease activity, but with differences in the cleavage behaviour with respect to the reactions observed with Mg2+. The existence of two separate activities (Mg2+-dependent and Mg2+-independent) for the production of DSB was confirmed by analysing the effect of EGTA, N-ethyl maleimide, ionic strength, and by preincubations of the nuclear extracts at different temperatures. The tissue distribution of both DSB-producing activities was also different. The in vitro system used in the present work may be a useful tool for studying the formation of DSB and for investigation of the mechanisms of DNA repair.