An in vitro assay was designed to study the import of radiolabeled ergosterol and cholesterol from unilamellar vesicles into isolated mitochondria of the yeast Saccharomyces cerevisiae. Supply of ergosterol to the mitochondrial surface was enhanced by a cytosolic fraction containing a lipid transfer protein, whereas no such additive to the assay was required for cholesterol transport. Both sterols reached the inner mitochondrial membrane. During import, they were detected in contact sites between the outer and the inner mitochondrial membrane supporting the idea, that these zones are sites of intramitochondrial lipid translocation. Transport of ergosterol between the outer and the inner mitochondrial membrane was not affected by addition of ATP, depletion of ATP caused by treatment of mitochondria with apyrase and oligomycin, and incubation with the uncoupler CCCP, indicating that this process is energy-independent.