The ansamycin antibiotic geldanamycin has frequently been used as an inhibitor of heat shock protein 90 (Hsp90), and this agent has been widely employed as a probe to examine the interactions of Hsp90 with endothelial nitric-oxide synthase. Geldanamycin contains a quinone group, which may participate in redox cycling. When geldanamycin was exposed to the flavin-containing enzyme cytochrome P-450 reductase, both semiquinone and superoxide (O(2)(*)(-)) radicals were detected using electron spin resonance. The treatment of endothelial cells with geldanamycin resulted in a dramatic increase in O(2)(*)(-) generation, which was independent of endothelial nitric-oxide synthase, because it was not inhibited by N-nitro-l-arginine methyl ester and also occurred in vascular smooth muscle cells. Diphenylene iodinium inhibited this increase in O(2)(*)(-) by 50%, suggesting that flavin-containing enzymes are involved in geldanamycin-induced O(2)(*)(-) generation. In the absence of cells, geldanamycin directly oxidized ascorbate, consumed oxygen, and produced O(2)(*)(-). Geldanamycin decreased the bioavailable nitric oxide generated by 3,4-dihydrodiazete 1,2-dioxide in smooth muscle cells by 50%, whereas pretreatment with superoxide dismutase inhibited the effect of geldanamycin. These findings demonstrate that geldanamycin generates O(2)(*)(-), which scavenges nitric oxide, leading to loss of its bioavailability. This effect is independent of the inhibition of Hsp90 and indicates that geldanamycin cannot be used as a specific inhibitor of Hsp90. In light of these findings, the studies using geldanamycin as an inhibitor of Hsp90 should be interpreted with caution.