Cardiolipin (CL), an anionic phospholipid constituting 20% of the inner mitochondrial membrane (IMM) of eukaryotes, stabilizes electron transport chain (ETC) complexes and is a signaling agent in the early stages of apoptosis. For apoptosis, CL moves from the inner to the outer leaflet of the IMM via a poorly understood mechanism. Relative to cylindrically shaped lipids like dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG), cone-shaped CL should prefer the concave surfaces of lipid bilayers. Using the fluorophore, 1,1,2,2-tetrakis[4-(2-trimethylammonioethoxy)phenyl]ethene, we have measured CL versus DOPG partitioning to the inner versus the outer leaflet of liposomes in mixed lipid systems with DOPC. DOPG shows no leaflet preference. However, CL has a 4:1 preference for the concave surface of the inner leaflet of liposomes. To further test the inner leaflet preference of CL, we show that cytochrome c binding to the outer convex surface of 20% CL/80% DOPC vesicles is strongly attenuated. Because the outer leaflet of intracristal regions of the IMM has a concave curvature, the preference of CL for concave surfaces may facilitate the transport of CL from the inner to the outer leaflet of the IMM needed for CL remodeling, the optimal functioning of the ETC, and signaling in the early stages of apoptosis.