Tricyclohexylhydroxytin, commonly known as Plictrana, inhibited Na+, K+-ATPase activity of rat brain synaptosomes in a concentration-dependent manner with median inhibitory concentration (IC-50) of 2 pM. Both K+-stimulated para-nitrophenylphosphatase and 13-HI-ouabain binding to synaptosomes were also inhibited by Plictran with IC-50 values of 11 and 30 pM, respectively. Altered pH and Na+, K+-ATPase activity curves demonstrated comparable inhibition in buffered neutral and alkaline pH ranges, and no inhibition was observed in acidic pH. The inhibition of Na+, K+-ATPase was independent of temperature. Kinetic studies of substrate (ATP) activation of Na+, K+-ATPase indicated uncompetitive inhibition. Results also showed noncompetitive inhibition for p-nitrophenylphosphate and uncompetitive inhibition for K+activations of p-nitrophenylphosphatase. Preincubation of synaptosomes with dithiothreitol, a sulfhydryl (SH) agent, resulted in the complete protection of Plictran inhibition of Na+, K+-ATPase, K+-para-nitrophenylphosphatase, and [3-HI-ouabain binding. The protection was specific and concentration dependent since cysteine and glutathione did not afford protection. These results indicate that Plictran inhibited Na+, K+-ATPase by interacting with dephosphorylation of the enzyme-phosphoryl complex and exerted a similar effect to that of SH-blocking agents.