Golgi apparatus from rat liver contain an ascorbate free radical oxidoreductase that oxidizes NADH at neutral pH with monodehydroascorbate as acceptor to generate a membrane potential. At pH 5.0, the reverse reaction occurs from NAD(+). The electron spin resonance signal of the ascorbate-free radical and its disappearance upon the addition of NADH (pH 7) or NAD(+) (pH 5.0) confirms monodehydroascorbate involvement. Location of monodehydroascorbate both external to and within Golgi apparatus compartments is suggested from energization provided by inward or outward flux of electrons across the Golgi apparatus membranes. The isolated membranes are sealed, oriented cytoplasmic side out and impermeable to NAD(+) and ascorbate. NAD(+) derived through the action of Golgi apparatus beta-NADP phosphohydrolase is simultaneously reduced to NADH with monodehydroascorbate present. The response of the NADH- (NAD(+)-) ascorbate free radical oxidoreductase system to pH in Golgi apparatus provides a simple regulatory mechanism to control vesicle acidification.