1. A method for the preparation of isolated mammary gland cells of the rat is described. 2. The procedure involves disaggregation of the tissue in a collagenase-hyaluronidase mixture and subsequent purification of the heterogeneous population of cells by centrifugation in discontinuous Ficoll-400 gradients; the preparation takes 60 minutes. The yield of cells is approximately 14%. 3. The cells as prepared have high rates of metabolism and synthetic capacity and exhibit metabolic characteristics comparable to intact tissue. 4. Measurements of the content of metabolic intermediates show cells to have, and retain, outstandingly high levels of ATP and to have an energy charge close to 0.9. Levels of other intermediates approximate to those found in the intact tissue. The level of glycolytic intermediates below the triose phosphate stage indicate the highly aerobic state of the cells. 5. The pattern and scale of glucose utilization, measured using specifically labelled glucose incorporation into 14CO2 and 14C-labelled lipid production, approximates closely in isolated cells at 5 and 20 mM glucose and in tissue slices at 20 mM glucose concentration. Mammary gland slices incubated with 5 mM glucose have a considerably lower rate of metabolism. Isolated cells exhibit a higher proportionate rate of glucose utilization by way of the pentose phosphate pathway. 6. The isolated cells are hormone responsive. Insulin increases the oxidation of glucose by the pentose phosphate pathway and stimulates lipid synthesis. Addition of progesterone and cortisone in vitro (10 muM) leads to a marked and rapid decrease in the rate of glucose oxidation and conversion to lipid.