Intact mycelia from wild-type Phycomyces blakesleeanus NRRL 1555(-) are capable to reduce the rate of chemical oxidation of extracellular ascorbate. Plasma membrane isolated from this fungus catalysed NADH oxidation using ferricyanide and ascorbate free radical (AFR) in a ratio of specific activities of 40:1. NADH-AFR reductase activity was stimulated by CoQ₆ and inhibited by chloroquine, the inhibition being partially reversed by CoQ₆. However, ferricyanide reductase activity was insensitive to both effectors. In addition, the extraction of CoQ from plasma membrane resulted in a lack of NADH-AFR reductase activity; addition of extra CoQ₆ to these extracted membranes restored about 85% the original activity. Ferricyanide reductase activity was unaffected under similar experimental conditions. NADH-AFR reductase activity can also be differentiated from ferricyanide reductase activity by its sensitivity to detergents: CHAPS and Triton X-100 inhibited AFR reductase, but have not effect on ferricyanide reductase activity. These results indicate that NADH-AFR reductase activity is a different enzymatic activity of previously described ferricyanide reductase activity, as well as the participation of CoQ in the AFR reduction by NADH-AFR reductase activity from Phycomyces blakesleeanus plasma membrane.