An HPLC assay for determination of ATPase activity was developed and validated. After stopping the enzyme reaction of the enzyme source (rat renal cortical basolateral membranes) with ATP, products derived from ATP were analyzed by two methods; HPLC determination of ADP derived from ATP, and colorimetry of inorganic phosphorus (Pi) released from ATP. This HPLC procedure was precise and linear over the range of protein amount of the enzyme source studied, and the intra-and inter-assay variations were lower than 10%. The values that were obtained by the two methods revealed a significant correlation. Also, even when the samples contained Pi or were contaminated with Pi, this HPLC method allowed determination of ATPase activity. In addition, when ouabain was used as an inhibitor, the HPLC method was found to be applicable for Na,K-ATPase determination. This indicated that this HPLC assay would enable determination of ATPases other than Na,K-ATPase, when other inhibitors are employed instead of ouabain.