Various drugs and chemicals break the DNA strand under ultraviolet irradiation. This study aimed to clarify the DNA-breaking activity (DBA) of serum from 39 patients with various photosensitivity disorders and that from eight normal subjects. A mixture of serum and circular plasmid DNA was exposed to longwave ultraviolet radiation, and the photoinduced cleavage of plasmid DNA was examined by electrophoretic analysis. DBA was found in serum from patients with erythropoietic protoporphyria (2 of 2), drug-induced photosensitivity (3 of 5), chronic actinic dermatitis (1 of 12) and hydroa vacciniforme (1 of 1). DBA was not found in serum from patients with porphyria cutanea tarda, collagen diseases with photosensitivity, papulovesicular light eruption or pellagra. The inhibition profile of DBA by active oxygen scavengers was different between afloqualone- and tetracycline-induced photosensitivity and chronic actinic dermatitis. The present method was useful for the detection of serum phototoxicity and the investigation of the pathomechanisms of photosensitivity.