Etoposide, a DNA topoisomerase II inhibitor, caused a concentration-dependent induction of apoptosis in immature thymocytes. Using a flow cytometric method to separate and quantify normal and apoptotic cells, etoposide-induced apoptosis was inhibited by cycloheximide and actinomycin D but not by zinc. Etoposide induced a marked cleavage of DNA into nucleosomal length fragments or multiples thereof, which was completely inhibited if the thymocytes were also incubated in the presence of zinc. Etoposide, alone, induced the classical ultrastructural features of apoptosis, but in the presence of zinc, the morphological pattern was markedly different and dominated by discrete clumps of condensed chromatin abutting the nuclear membrane. These latter changes resemble those described as the earliest changes in apoptosis. These results support the hypothesis that, in the induction of apoptosis, critical alterations in nuclear chromatin occur prior to endonuclease cleavage of DNA into nucleosomal fragments.