Cytochrome oxidase, an enzyme containing six different subunits, has been shown to span the inner mitochrondrial membrane. The arrangement of the subunits within the membrane is unknown. Wh have specifically labeled the 25 000 molecular weight subunit with a spin-label derivative of N-ethylmaleimide, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (NEM-SL(5)). NEM-SL(5)-lebeled cytochrome oxidase can be incorporated into phospholipid membranes to form coupled vesicles of the Hinkle, Kim & Racker ((1972) Jriol. Chem; 247, 1338-1399) type. The resonance spectrum of NEM-SL(5) is similar in both soluble and vesicular cytochrome oxidase. Since ascorbate has been shown to reduce only spin label that is exposed to the exterior surface of a closed vesicle, we have used ascorbate to determine the NEM-SL(5)-binding site in the coupled vesicles; NEM-SL(5)-labeled cytochrome oxidase vesicles are reduced by 10 mM ascorbate with tau 1/2 of 1 min at 22 degrees C; The rate of reduction is relatively independent of temperature. We conclude that (1) cytochrome oxidase is unidirectionally or preferentially oriented in the vesicle membrane, and (2) the NEM-SL(5)-binding site on the 25 000 molecular weight subunit is exposed to the external aqueous medium.