3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) is a photoactivatable carbene precursor designed to label selectively the hydrophobic core of membranes. We have used this reagent to obtain information on the topological organization of the membrane-embedded subunits of F1F0 ATP synthase from Escherichia coli. The study included [125I]TID labeling of F0 subunits in different structural (conformational) states and Edman degradations of the labeled polypeptides in order to assign the covalently bound radioactivity to individual amino acid residues. Released phenylthiohydantoin amino acids were analyzed by thin-layer chromatography, and the radioactive derivatives were visualized by autoradiography. The data suggest that labeling patterns can be correlated in a meaningful manner with reagent accessibility and hence with protein-lipid contact. Subunit b appears to be anchored to the membrane by a short N-terminal segment. As almost all of the amino acids of this part are accessible to the reagent, it is inferred that this segment has little interaction with the other subunits. In contrast, in the two segments of subunit c that were labeled with [125I]TID, only certain amino acids reacted with the label. The pattern of these labeled residues is compatible with that of tightly packed alpha-helices.