Mechanisms which determine sodium and potassium content and volume of rat thymic and human chronic lymphocytic leukemia (CLL) lymphocytes have been studied. The deleterious effect of cell isolation on monovalent cation content was proven by comparing thymus sodium and potassium concentration to that of thymocytes prepared from autologous hemithymus. In vivo distribution ratios of sodium‐24 and potassium‐42 between thymus water and plasma water were very similar to the distribution ratios of non‐radioactive isotopes (sodium‐23 and potassium‐39). The similar lymphocyte: thymocyte ratio of (a) cell volume (1.48), (b) cell sodium plus potassium (1.47) and (c) cell water (1.50) demonstrated the close correlation of lymphocyte volume with monovalent cation content and water content. Steady‐state CLL lymphocyte sodium (32 ± 1.9 mM) and potassium (131 ± 5.1 mM) and thymocyte sodium (31 ± 1.2 mM) and potassium (136 ± 3.9 mM) were similar; however, these steady‐state levels were maintained by quantitatively different membrane functions. Radiopotassium and radiosodium uptake by thymocytes was more rapid than by CLL lymphocytes. Ouabain‐sensitive potassium influx was 2.4 times greater in thymic (8.70 ± 2.28 mmoles/cm2/min × 10−8) than in CLL (3.24 ± 0.45 mmoles/cm2/min × 10−8) lymphocytes. Potassium exodus was also slower in CLL lymphocytes as compared to thymocytes. Ouabain‐sensitive sodium accumulation and ouabain‐insensitive sodium accumulation were also slower in CLL lymphocytes than in rat thymocytes. Half‐maximal ouabain inhibition of sodium entry was 7.5 × 10−3 M in thymic and CLL lymphocytes. The inhibitory effect of ouabain on sodium and potassium transport was easily reversible. Oligomycin inhibited ouabain‐sensitive potassium accumulation in both lymphocyte types. Four lines of evidence indicate the presence in the lymphocyte of a system of leaks and pumps, the latter subserved by a ouabain and oligomycin‐sensitive (sodium‐potassium) ATPase: (a) steady‐state monovalent cation gradient (K ∼ 20:1, Na ∼ 5:1), (b) the inability to maintain normal sodium and potassium gradients at cold temperature and in the presence of ouabain, (c) the effect of ouabain and oligomycin on active potassium influx and (d) the restitution of steady‐state sodium and potassium concentration after cell isolation, ouabain treatment and cold exposure. CLL lymphocytes as compared to rat thymocytes have a decreased rate of ouabain‐insensitive sodium uptake and potassium exodus requiring a reduced rate of active sodium extrusion and potassium accumulation to maintain steady‐state cation content. Ouabain‐sensitive ATPase is difficult to locate in lymphocytes in vitro possibly because it comprises a very small proportion of membrane ATPase since magnesium activated ecto‐ATPase in intact lymphocytes is 1500 to 2500 times that of the intact erythrocyte. The inhibition by ouabain of blast transformation, mitosis, amino acid accumulation and nucleic acid synthesis in vitro, may reflect the importance of ouabain‐sensitive ATPase and monovalent cation transport in the function of lymphoid cells.