A continuous-flow method was developed for determining the stoichiometry of the gastric proton pump H,K-ATPase (EC 3.6.1.36) in its hydrolysis of ATP and translocation of H+ and the K+ congener 86Rb+. H,K-ATPase-containing vesicles which had been isolated from pig gastric mucosa were incubated at 37 degrees C for 2 h in 150 mM 86RbCl, 0.5 mM ethylenebis(oxyethylenenitrilo)tetra-acetic acid and 3 mM 2-(N-morpholino)ethane sulphonic acid (Mes) adjusted to pH 6.1 with Tris, and then applied onto a 0.45 micron pore size cellulose acetate filter. The immobilized vesicles were superfused with 0.15 mM Mes/Tris buffer, pH 6.1, containing 150 mM choline chloride and 0.2 mM MgCl2. After changing to a medium containing 0.1 mM ATP, the amounts and rates of H+ uptake, 86Rb+ efflux and ATP hydrolysis were measured. The initial ratio of Rb+ transported to ATP hydrolysed gave values of 0.96 +/- 0.26 (mean +/- SD, n = 28). The initial ratio of ATP-dependent Rb+ efflux to H+ uptake gave values of 0.92 +/- 0.28 (mean +/- SD, n = 28). The Mg-ATPase activity was measured in vesicles which had been incubated with choline chloride instead of RbCl. This activity was 15.8 +/- 8.7% (mean +/- SD) of the total ATPase activity in the initial fractions used for calculation of the stoichiometry. It is argued that this Mg-ATPase may be an intrinsic activity of the H,K-ATPase and that the relation between these activities is dependent on the amount of K+ (or Rb+) present in the assay.(ABSTRACT TRUNCATED AT 250 WORDS)