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Nanoparticulate phospholipid bilayer disks were assembled from phospholipid and a class of amphipathic helical proteins termed membrane scaffold proteins (MSP). Several different MSPs were produced in high yield using a synthetic gene and a heterologous expression system and purified to homogeneity by a one-step purification. The self-assembly process begins with a mixture of the phospholipid and MSP in the presence of a detergent. Upon removal of detergent, 10-nm diameter particles form containing either saturated or unsaturated phospholipid. The ratio of components in the initial mixture was found to be crucial for formation of a monodisperse population of nanoparticles. Exploration of the phase diagram of the lamellar to phospholipid−detergent mixed micelle transition reveals that self-assembly proceeds from the mixed micellar phase. In this case a homogeneous and monodisperse population is …