The membrane-restricted photoactivatable carbene generator 3-(trifluoromethyl)-3-(m-[125I]-iodophenyl)diazirine [Brunner, J., & Semenza, G. (1981) Biochemistry 20, 7174-7182] was used to label the subunits of the membrane attack complex of complement (C5b-9). C5b-9 complexes either were assembled from serum on erythrocyte membranes or were reconstituted from purified components on liposomes. After irradiation, most of the probe is bound to C9 independent of the membrane system used, indicating that the wall of the transmembrane channel is predominantly composed of C9. No difference was observed whether polymerized C9 was in the tubular or nontubular form [Podack, E. R., & Tschopp, J. (1983) J. Biol. Chem. 257, 15204-15212], showing that tubule closure is not essential for successful lipid insertion. The same label distribution between the two forms of polymerized C9 was obtained by analyzing zinc-polymerized C9 in the absence of C5b-8. Since the photoreactive probe reacted with at least two distinct polypeptide segments within C9, lipid interaction does not occur via a single segment of hydrophobic amino acids.