A buoyant density gradient procedure for mammalian cell separation is described. Gradients of Ficoll and Hypaque in balanced salt solution were used in a zonal rotor to separate distinct leukocyte populations from human peripheral blood and bone marrow. This method possesses the advantages of uniform osmolarity, density determination by refractometry, ease of preparation of gradient solutions, large sample capacity, and low viscosity with consequent ease of sterilization. Granulocyte-monocyte colony-forming units (CFU-C) from blood and bone marrow banded as a homogeneous peak with a density of 1.056 g cm-3 on steep gradients. Recovery of CFU-C was 36% with a 7- to 9-fold enrichment on shallow gradients. Both fractionated and unfractionated peripheral blood cells were stimulated by phytohemagglutinin (PHA), Concanavalin A (Con A), and allogeneic lymphocytes. In contrast, cells from unfractionated bone marrow and cells from the light density fraction of marrow were inhibited by Con A and responded variably to PHA.