The nature of G2-arrest was studied in Chinese hamster ovary cells after exposure to 4′ -demethyl-epipodophyllotoxin 9-(4,6-O-2-thenylidene-β-d-glycopyranoside (VM26), 1,3bis( 2-chloroethyl)-1-nitrosourea, 1,3-cis(2-chloroethyl)-1-nitrosourea, cis-4-[[[2-chloroethyl]-nitrosoamino] carbonyl] amino]cyclohexane carboxylic acid, or neocarzinostatin. To determine whether this G2-arrest was due to a metabolic block or to damage to the genetic material, we employed the phenomenon of premature chromosome condensation to visualize the chromosomes of the G2-blocked cells by fusing them with mitotic cells. The prematurely condensed chromosomes (PCC) of the treated cells were scored for their position in the cell cycle and the extent of chromosomal damage in the G2-PCC. The data revealed a significant enrichment of the G2-fraction in all the treatments. Most of the G2-PCC from the treated cells were extensively damaged, showing 10 or more breaks and exchanges per cell. These studies clearly indicated that the treated cells accumulated in G2-phase because of their failure to progress into mitosis as a direct or indirect result of the extensive damage to the chromosomes.