Internucleosomal DNA fragmentation is often regarded as the biochemical hallmark of apoptosis and can be reproduced in vitro in rat liver nuclei. In this study we demonstrate that DNA is initially cleaved into > or = 700, 200-250, and 30-50 kilobase pair (kbp) fragments via a Mg(2+)-dependent, multistep process which can be potentiated by Ca2+. The subsequent internucleosomal cleavage requires both Ca2+ and Mg2+. Furthermore, we show that the heavy metals Cd2+ and Hg2+, dichloroisocoumarin (a general serine protease inhibitor), and N-ethyl maleimide (NEM, a specific thiol reagent) are potent inhibitors of both the Mg(2+)- and Ca2+/Mg(2+)-stimulated DNA fragmentation. In contrast, two other serine protease inhibitors, N-alpha-tosyl-L-lysine chloromethylketone and N-tosyl-L-phenylalanine chloromethylketone are weak and ineffective, respectively, as inhibitors of DNA cleavage. Increasing inhibition of DNA cleavage is accompanied by a shift in the size of the cleaved DNA fragments, which increases from mono- + oligo-nucleosomes-->30-50 kbp-->200-300 kbp--> > or = 700 kbp-->intact DNA. Dithiothreitol, a dithiol, blocks NEM and dichloroisocoumarin inhibition, and since Cd2+ and Hg2+ are also potent--SH blocking agents it is proposed that a critical thiol is involved in the cleavage of DNA into both large kbp fragments and oligonucleosomal-sized fragments.