Cytochrome P450scc can be reconstituted successfully into large unilamellar phospholipid vesicles by a combined octylglucoside dialysis/adsorption method. Freeze-fracture electron microscopy was used to analyze the morphology, distribution, and protein topology of the cytochrome P450scc vesicles in dependence on lipid composition. Particles were observed only in close contact to the vesicle surface, probably representing tightly associated cytochrome P450scc at the outer vesicle surface. In cytochrome P450scc vesicles similar in lipid composition to the inner membrane of bovine mitochondria direct evidence by freeze-fracturing was found for a specific cytochrome P450scc-induced aggregation of the vesicles. The vesicle aggregation critically depends on the content of the specific mitochondrial membrane constituent cardiolipin. The aggregation and thus the intervesicular contacts were observed to be inhibited by both addition of anti-cytochrome P450scc IgG and adrenodoxin. Enzymatic reduction of cytochrome P450scc in the liposomal membrane by its electron transfer partners completely indicates an asymmetrical localization in/at the outer side of the bilayer membrane. It is suggested that vesiculation of the inner mitochondrial membrane may be a consequence of the characteristic cardiolipin dependent cytochrome P450scc membrane topology: the cardiolipin binding, peripheral, non-bilayer-spanning integration as an oligomer in the outer leaflet of the membrane may play a role in the dynamics of formation and dissociation of intramitochondrial vesicles with a functional importance for steroidogenesis. (C) 1994 Academic Press, Inc.