Our aim was to set up and validate a reproducible method to study ATPase family on erythrocyte membranes. We compared several methods for erythrocyte washing and hemolysis and succeeded in preparing completely hemoglobin-free membrane ghosts still bearing intact ATPases. We compared the conventional incubation procedure with the coupled enzyme assay to measure Na-K-Mg, Ca-Mg and Mg ATPase on the membranes. A significant difference was constantly observed between the results by these methods, the values by the incubation procedure being 28, 57 and 58% of the respective values obtained by the linked enzyme assay. By adopting this last one, we obtained uniform and reproducible results in 31 healthy subjects. The following activities of the measured pumps resulted: Na-K-Mg ATPase 0.026 +/- 0.007, mean +/- SD; Ca-Mg ATPase 0.030 +/- 0.010, and Mg ATPase 0.017 +/- 0.003 U/mg protein, respectively. Finally, we investigated the effect of membrane storage time and temperature on ATPase results.