The distribution of cardiolipin across the inner mitochondrial membrane was 
directly determined by using the ability of the fluorescent dye 
10-N-nonyl-3,6-bis(dimethylamino)acridine (10-N-nonyl acridine orange) to form 
dimers when it interacts with the diacidic phospholipid. Two independent methods 
were employed: (a) a spectrophotometric measurement of 10-N-nonyl acridine 
orange binding to isolated rat liver mitochondria, mitoplasts and inside-out 
submitochondrial particles, and (b) a flow-cytometric analysis of specific red 
fluorescence, emitted when two dye molecules are bound to one membrane 
cardiolipin; the stoichiometry of 10-N-nonyl acridine orange binding to 
phosphatidylserine and phosphatidylinositol, 1 mol dye/mol phospholipid, 
prevented dye dimerisation and subsequent red-fluorescence appearance. 57% total 
cardiolipin was present in the outer leaflets of inner membranes of isolated 
organelles, a distribution confirmed by saturation measurements for mitoplasts 
and inside-out submitochondrial particles. The same asymmetry was directly 
observed in situ with mitochondrial membranes of quiescent L1210 cells, and with 
mitochondrial membranes of respiring yeasts. Nevertheless, alterations in ATP 
synthesis and inhibition of mitochondrial protein synthesis revealed that 
cardiolipin distribution was apparently tightly correlated with mitochondrial 
membrane assembly and activity.