The effect of external ATP on the steady-state levels of water and electrolytes in isolated renal tubules of healthy adult rabbits was studied: 1. 1. In the absence of external Ca 2+, ATP produced an increase of tissue Na +, Cl −, and Ca 2+, and a loss of K +, at all pH values tested (pH 6.2 to 8.2); only at pH 7.2 was a marked ATP-induced cellular swelling observed. No such effects of ATP were found in the presence of 2.5 mM [Ca 2+] 0. 2. 2. The swelling effect of ATP was not affected by 0.1 mM and 0.5 mM ouabain or the absence of Na + (Li + saline) and was in part reversed by raising [Ca 2+] 0 to 2.5 mM. Increased Mg 2+ (5.5 mM final concn) could replace Ca 2+ in preventing the effect of external ATP. 3. 3. ADP and GTP did not produce changes of cell volume under conditions where ATP was effective. 4. 4. Isolated kidney tubules were found to hydrolyse added ATP or other triphosphonucleotides. The rate of hydrolysis was increased by raising the medium pH from 6.2 (0.13 μmole P 1/mg protein per 10 min) to 8.2 (0.24 μmole P i/mg protein per 10 min) and was to some extent inhibited by Ca 2+. 5. 5. A soluble protein with a nucleotidase specificity, activated by Mg 2+ or Ca 2+, was isolated from the tubules. At pH 7.2 this protein was precipitated from solution by the addition of ATP and Ca 2+; this phenomenon was found to be specific for ATP. 6. 6. It is suggested that external ATP affects the water and ionic contents of the cells of kidney tubules by an interaction with the cell membrane and Ca 2+, thus involving the physical (contractile?) properties of the membrane structure and/or membrane permeability.