A neutral filter elution assay was used to determine if the post-replicational formation and repair of DNA double-strand breaks (DSB) occurs in u.v.-irradiated human cells. Excision-deficient XP12 cells were pulse-labeled with [3H]thymidine after u.v. irradiation (1.5–3 J/m2), and the nascent DNA was followed during repair incubation. With increasing u.v. radiation fluences, an increasing fraction of DNA was eluted at a fast rate, indicating that DSB were produced. The maximum yield of DSB was observed after about 24 h of post-irradiation incubation at 37°C. Similar results were also obtained with repair-proficient VA13 cells when irradiated at much higher fluences (7.5–15 J/m2). It is concluded that, at the u.v. radiation fluences used in this work, the DSB produced in u.v.-irradiated human cells are the result of post-replication repair events, and at incubation times >24 h some of these DSB are repaired.