This laboratory has recently reported the occurrence of DNA nicking at the onset of terminal skeletal myogenesis by using the technique of in situ nick translation (Dawson and Lough: Dev. Bio/., 1271362-367, 1988). Because 1 -beta-D-arabinofuranosylcytosine (araC), a cytocidal agent that is routinely used to remove dividing fibroblasts from myogenic cultures, inhibits DNA repair, it was of interest to determine whether araC treatment resulted in an accumulation of the endogenously created nicks. Thus, we have assessed the accumulation of DNA nicks in myotube cells during a 20 hour araC treatment period at the onset of terminal myogenesis (44-64 hours in vitro) by using three techniques: alkaline sucrose gradient density centrifugation, kinetic in situ nick translation, and cellular in situ nick translation. Although alkaline sucrose gradient centrifugation revealed no detectable nicking after 20 hours, kinetic in situ nick translation analysis revealed subtle but significant increases in DNA nicks caused by araC within 7 hours of drug application, and a 1 .S-fold increase in DNA repair sites after 20 hours of drug treatment. That these observations reflected nicking specifically in myotube nuclei was determined by irnrnunocytochemical localization of nicked sites after repair with a biotinylated nucleotide analog (biotin-I 1 -dUTP). The effects of araC were only incompletely reversible, whether or not the drug was removed from the cultures, within 2 days of the treatment period. These results indicate that araC inhibits the repair of endogenously created nicks in the DNA of non-dividing, terminally differentiating myotube cells, and provide further evidence for DNA nicking and repair as a feature of terminal myogenic differentiation.