Inactive forms of endo-exonuclease, activated in vitro by treatment with trypsin, have been identified in human leukaemic CEM and MOLT-4 cells. They comprise over 95% of the total single-strand DNase activity in nuclei and are mainly bound to chromatin and the nuclear matrix. The activated enzyme had Mg2+(Mn2+)-dependent, Ca(2+)-stimulated activities with single- and double-strand DNAs and RNA (polyriboadenylic acid) and other properties characteristic of endo-exonucleases previously described. At least twice as much inactive endo-exonuclease has also been localised in extranuclear compartments of CEM and MOLT-4 cells, 85% bound to the membranes of the endoplasmic reticulum and 15% free in the cytosol. The soluble cytosolic trypsin-activatable endo-exonuclease was immunoprecipitated by antibodies raised independently to both Neurospora and monkey CV-1 cell endo-exonucleases. The free and bound enzymes of both nuclear and extranuclear compartments also cross-reacted on immunoblots with the antibody raised to Neurospora endo-exonuclease to reveal multiple polypeptides ranging in size from 18 to 145 kDa, many of which exhibited activity on DNA gels. The major species bound to the chromatin/matrix were in the 55-63 kDa range. Limited proteolysis of the large polypeptides to those of 18 to 46 kDa accompanied spontaneous chromatin DNA fragmentation to form DNA "ladders' in an isolated nuclei/cytosol system. When the leukaemic cells were treated in culture with either etoposide or podophyllotoxin to induce apoptosis, the largest polypeptides disappeared and smaller endo-exonuclease-related polypeptides of 18 to 46 kDa were detected in the nuclear extracts. The appearance of these polypeptides also correlated with extensive chromatin DNA fragmentation. In addition, there were correlations between the depletion of the major 55-63 kDa species bound to the membranes of the endoplasmic reticulum, depletion of the extranuclear trypsin-activatable activity and the onset and extent of chromatin DNA fragmentation in both cell lines. The extranuclear 55-63 kDa species may be precursors of the chromatin/matrix bound endo-exonuclease. The results indicate that endo-exonuclease plays a role in chromatin DNA degradation in mammalian cells during apoptosis.