Abstract—Upon irradiation with ultraviolet light the isoflavonoid phytoalexins phaseollin, 3,6a, 9‐trihydroxypterocarpan, glyceollin, tuberosin and pisatin, but not medicarpin, brought about inactivation ofglucose–6‐phosphate dehydrogenase in anin vitroassay system. Photoinactivation of the enzyme by photoactivated pisatin in air‐saturated solutions was hardly affected by singlet oxygen quenchers such as NaN3, bovine serum albumin, histidine or methionine. Neither addition of the hydroxyl radical scavengers mannitol, Na‐benzoate and ethanol nor the presence of catalase or supcroxide dismutase protected the enzyme against photoinactivation, suggesting that OH, H2O2and O2are not the reactive oxygen species involved. However, the free radical scavenger S‐(2‐amino‐ethyl)isothiouronium bromide hydrobromide (AET) protected the enzyme against inactivation by photoactivated pisatin. Direct evidence for the generation of free radicals was obtained by ESR measurements of solutions of phaseollin, pisatin and medicarpin in hexane irradiated with ultraviolet light in the presence or absence of O2. Phaseollin produced the most stable free radicals, whereas medicarpin hardly gave rise to free radical formation; pisatin took a somewhat intermediate position by producing a strong ESR signal which, however, decayed rather quickly. Photodegradation of all phytoalexins, except for medicarpin, was accompanied with loss of fungitoxicity, as shown in thin‐layer chromatographic bioassays, and formation of new products.These results indicate free radical formation as the causative process for photoinactivation of enzymes by photoactivated isofla