N-Diazoacetylglycine amide, a diazochetoalkane, has been studied in vitro for DNA damage and repair in cells of a cloned subline from a BALB/c mouse. To our present knowledge, none of these compounds have been investigated for such activities. At nontoxic levels, a prolonged dose-dependent unscheduled DNA synthesis was observed by autoradiography. DNA damage was studied by sedimentation through alkaline sucrose gradients after the cells were lysed on the gradients. Treatment of the cells for 1 hr with nontoxic doses of N-diazoacetylglycine amide resulted in slower sedimentation of DNA. The number of single-strand breaks appeared rather linearly dose dependent for a large range of concentrations. Breaks were at their maximum after 1 hr of treatment, and no further increase in the number of breaks was seen. Some repair of the breaks probably occurs, but repair was sluggish even 68 hr after treatment. A significant part of the breaks was observed after incubation at 4 degrees in an ethylenediaminetetraacetate hypotonic solution. This seems to indicate that the compound does not require metabolic activation. Nontoxic doses of N-diazoacetylglycine amide and other similar derivatives exert mutagenic and carcinogenic activities. The presence of DNA damage and the difficulty in its repair at such doses could be related to both of these biological properties.