Some fluoroquinolone antibiotics (FQs) become toxic and mutagenic upon exposure to ultraviolet radiation (UV). Topoisomerase inhibition has been proposed as one possible mechanism involved in this photochemical genotoxicity. To study this reaction, inhibition of the human topoisomerase IIalpha enzyme by four FQs varying in photochemical genotoxic potency (Bay y3118 [y3118] > Lomefloxacin [Lmx] > Ciprofloxacin [Cpx] > Moxifloxacin [Mox]) was measured in vitro in the presence of UVA irradiation. None of the FQs inhibited topoisomerase IIalpha in the absence of irradiation. In contrast, with irradiation at 365 nm, the potent photochemically genotoxic y3118 produced strong inhibition of the enzyme by 15% and Cpx caused a weak 5% inhibition, but the more photochemically genotoxic Lmx only showed a transient inhibitory effect at one concentration and one irradiation dose. The photostable Mox had no effect with irradiation. Topoisomerase IIalpha inhibition by y3118 only occurred when the FQ, DNA, and enzyme were simultaneously present in the UVA-irradiated reaction mixture and was abolished in the absence of ATP, indicating the possible formation of a ternary structure. The y3118 photochemical topoisomerase inhibition correlated with the increased irradiation-mediated binding of radiolabeled FQ to DNA:topoisomerase complexes and was irreversible, like that of the topoisomerase poison, etoposide, without irradiation. The inhibitory effect of photoactivated y3118 on topoisomerase IIalpha was also observed in the presence of the antioxidant TEMPO, indicating that reactive oxygen species were not involved in the inhibition. These observations demonstrate that some but not all photochemically genotoxic FQs inhibit human topoisomerase IIalpha, possibly by UV-induced affinity of FQs to DNA:topoisomerase complexes.