An assay for activities of erythrocyte membrane-bound ATPases adapted from the autoanalyzer method of Raess and Vincenzi (1980a) is described in detail. Mg2+ ATPase, Na+/K(+)-ATPase, Ca2+ ATPase, and calmodulin- (CaM) activated Ca2+ ATPases were determined in microtiter plates. Total volume was 100 microL. Ten microliters of 0.75 mg/mL of red blood cells membranes were added to appropriate buffer in microtiter plates. Plates were preincubated at 37 degrees C for 10 min, reactions were started by the addition of ATP, and plates were incubated for an additional 60 min at 37 degrees C. Reactions were stopped by sodium dodecyl sulfate (SDS). Inorganic phosphate (P(i)) was measured by modifications of the method by Fiske and Subbarow, (Fiske and Subbarow, 1925) in the same plate using a plate reader. The P(i) assay range was between 0 and 250 nm/mL. Results obtained for intraassay precision, (n = 7) are as follows: Mg2+ ATPase = 4.39 +/- 0.25 (5.7% CV); Na+/K(+)-ATPase = 7.33 +/- 0.40 (5.4% CV); Ca2+ ATPase = 15.86 +/- 0.76 (4.8% CV); and CaM-activated Ca2+ ATPase = 74.12 +/- 2.34 (3.2% CV) (nmole P(i)/mg protein/min.). This is a rapid, simple, and nonisotopic method for the determination of membrane-bound ATPases activities. All steps are performed in the same microtiter plate, thus reducing handling and associated errors.