The purpose of this work was to isolate thymocyte plasma membranes at high yield and purity to study specific surface molecules in their structural context. A procedure was developed in which 92-95% of the cells were disrupted by homogenization in a dense viscous medium, while nuclei remained intact. Differential centrifugation of the homogenate was avoided; instead, only a brief (2 h) centrifugation at equilibrium- density of membrane components was used. Five fractions were obtained, three by flotation. Membrane-bound enzymatic activities indicated a 60- 80% yield of plasma membranes in the three floated membrane fractions, which comprised 1.6% of the homogenate protein. Enrichment factors for three ectoenzymes, alkaline phosphatase, gamma-glutamyltransferase, and ouabain-sensitive adenosine triphosphatase were respectively, 70-74, and 40-50 in the two lightest fractions. Nuclear membranes were then isolated from the remaining whole nuclei and were found to be enriched in esterase and NADH-cytochrome c reductase. Plasma membranes and light nuclear membranes appeared as pure unit-membrane vesicles in thin sections and freeze-etching electron microscopy. Some aggregation of intramembranous particles occurred in plasma membrane vesicles.