We describe a method for the isolation of plasma membranes and other organelles from the lymphocytes of pig mesenteric lymph nodes, as well as from calf mediastinal nodes. The cells were disrupted by nitrogen cavitation leaving the nuclei intact. Nuclei, the large granule fraction and microsomes consisting of vesicles derived from plasma membrane and endoplasmic reticulum were separated by differential centrifugation. Plasma membranes were separated from the fragmented endoplasmic reticulum by equilibrium density ultracentrifugation in buffered dextran solutions. All fractions were characterized using marker enzymes and by their chemical composition. The microsomal fraction seemed to be free of cell constituents other than plasma membranes and endoplasmic reticulum. The yield of plasma membranes was 1.7 % (protein content relative to whole cell homogenate). Specific activities of 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5), a widely used plasma membrane marker, were 125 nmoles·(mg protein) −1·min −1 being enriched by a factor of 25 as compared with whole cell homogenate. The cholesterol content of plasma membranes was 751 nmoles/mg protein (homogenate = 104 nmoles/mg protein) and the phospholipid content 727 nmoles/mg protein (homogenate = 90.3 nmoles/mg protein). The cholesterol: phospholipid molar ratio was 1.03. Lysolecithin acyltransferase (acyl-CoA: 1-acyl- sn-glycero-3-phosphorylcholine- O-acyltransferase, EC 2.3.1.?.) showed specific activities between 10 to 12 nmoles·(mg protein) −1·min −1 in the plasma membrane compared to 3–4 nmoles·(mg protein) −1·min −1 in endoplasmic reticulum and thus appears to be a plasma membrane component in lymphocytes.