Thymidylate synthase-negative mutant mouse cells starved of thymidine or their parental FM3A cells treated with 5-fluoro-2'-deoxyuridine produced DNA fragments ranging from 50 to 200 kilobase pairs with a peak at 100 kb in length as determined by pulsed-field agarose gel electrophoresis. Accumulation of the DNA fragments following such thymidylate stress was time-dependent but their size distribution did not change in either case. Regions of the chromosomal DNA breaks seemed to be restricted to those where DNA replication was in progress as shown by pulse-labeling of the DNA synthesis. Emetine, an inhibitor of protein synthesis, blocked the accumulation of the DNA fragments when present during thymidylate stress. Cell-free extracts prepared from the thymidylate-stressed cells derived by either of the above means were capable of degrading DNA in chromatins prepared from normally growing cells in vitro. The resulting DNA fragments were similar but with a somewhat broader size distribution compared to those produced in vivo. The broader distribution of the fragments produced in the in vitro reaction became closer to the pattern obtained in vivo when ATP and 4 deoxyribonucleotides were added to the reaction.