DNA repair plays an essential role in maintaining genomic stability, and can be assessed by various comet assay-based approaches, including the cellular repair assay and the in vitro repair assay. In the cellular repair assay, cells are challenged with a DNA-damaging compound and DNA damage removal over time is assessed. In the in vitro repair assay, an early step in the repair process is assessed as the ability of a cellular extract to recognize and incise damaged DNA in substrate nucleoids from cells treated with a DNA-damaging compound. Our direct comparison of both assays in eight cell lines and human peripheral blood lymphocytes indicated no significant relationship between these DNA repair assays (R2= 0.084,P= 0.52). The DNA incision activity of test cells measured with the in vitro repair assay correlated with the background level of DNA damage in the untreated test cells (R2= 0.621,P= 0.012). When extracts were prepared from cells exposed to DNA-damaging agents (10 mM KBrO3or 1 µM Ro 19–8022 plus light), the incision activity was significantly increased, which is in line with the notion that base excision repair is inducible. The data presented suggest that the two assays do not measure the same endpoint of DNA repair and should be considered as complementary.