Treatment of hog gastric microsomes with the sulfhydryl reagent, thimerosal (ethylmercurithiosalicylate), produced differential effects on the K +-ATPase and the K +-stimulated p-nitrophenylphosphatase activities. For example, exposure to 2 mM thimerosal for 3 min severely reduced the activity of K +-stimulated ATPase, while K +- p-nitrophenylphosphatase activity was enhanced 2- to 3-fold. Higher concentration of thimerosal, or longer incubation times, also led to inhibition of K +- p-nitrophenylphosphatase. The activated state of p-nitrophenylphosphatase could be sustained by a 20-fold, or greater, dilution of treated membranes, and could be reversed by reduction of membrane SH groups by exogenous thiols. Significant activation of K +- p-nitrophenylphosphatase was not produced by p-chloromercuribenzene sulfonate, p-chloromercuribenzoate or mersalyl; however, ethyl mercuric chloride had qualitatively similar activity effects as thimerosal. Kinetics of K +- p-nitrophenylphosphatase for thimerosal-treated membranes were altered as follows: V increased; K m for p-nitrophenylphosphate unchanged for K a for K + increased. ATP, which is a potent inhibitor of K +- p-nitrophenylphosphatase activity in native membranes ( K I ≈ 200 μM). These data suggest that there are multiple SH groups which differentially influence the gastric K +-stimulated ATPase activity. Defined treatments with thimerosal are interpreted as an uncoupling of the K +-stimulated phosphatase component of the enzyme (for which p-nitrophenylphosphatase is a presumed model reaction). Such differential modifications can be usefully applied to the study of partial reactions of the enzyme and their specific role in the related H +-transport reaction.