Abstract The nature of DNA interactions with photoactivated gilvocarcin V has been analyzed at the gene level in both rRNA and dihydrofolate reductase genes of human fibroblasts, utilizing a modified Southern hybridization technique. Neither interstrand DNA crosslinking nor RNA linkage to DNA was detected. However, we consistently observed in both genes retarded DNA bands appearing in a dose-dependent fashion following exposure to photoactivated gilvocarcin V. These retarded bands were enhanced when genomic DNA was prepared without proteinase K treatment, suggesting involvement of protein in this DNA interaction. Because these bands disappear following proteinase K treatment, it is probable that photoactivated gilvocarcin V induces DNA-protein crosslinking.