A recently derived in vitro chemiluminescence assay (Salles et al. [1995] Anal. Biochem., 232, 37-42) has been used to investigate the effects of a panel of twenty-two anticancer drugs and certain antibiotics on the excision repair activity of cell-free extracts from the human cell line, HeLa. This methodology, termed the 3D (Damaged DNA Detection) assay, based on the in vitro excision repair assay previously developed (Wood et al. [1988] Cell, 53, 97-106) has provided data indicating definite in vitro inhibition of DNA repair by actinomycin D, aphidicolin, doxorubicin, distamycin A and mithramycin A. This assay therefore offers the potential for identifying agents with the ability to inhibit DNA repair.