The in vitro incorporation of a well-characterized integral protein cytochrome b5 into membranes of various subcellular organeUes was investigated by biochemical and immunochemical methods. Microsomes, peroxisomes, and outer mitochondrial membranes, all containing endogenous cytochrome b5, incorporated large amounts of the hemoprotein in such a way that it was reducible by an inherent NADH cytochrome bs reductase. Lysosomal membranes did not incorporate cytochrome bs. Inner mitochondrial and Golgi membranes, which do not naturally contain cytochrome bs, bound it in vitro but it was not reduced in the presence of NADH. These results show some discrepancies between the natural localization and the in vitro binding of cytochrome bs. They confirm one aspect of the fluid membrane theory and bring new elements to our understanding of the maintenance of the specific features of the membranes of subcellular organelles with respect to the cell dynamism.