The mechanisms of inhibition of rat brain Na+-K+-ATPase by cadmium chloride (CdCl2) and methylmercuric chloride (CH3HgCl) were studied in vitro by assessing the effects of these heavy metals on this enzyme and associated component parameters. Both the heavy metals significantly inhibited the overall Na+-K+-ATPase in a concentration-dependent manner with an estimated median inhibitory concentration (IC-50) of 3.2 X 10(-5) M for CdCl2 and 6 X 10(-6) M for CH3HgCl. Protection of enzyme against heavy metal inhibition by 5 X 10(-5) M to 1 X 10(-4) M dithiothreitol (DTT) and glutathione (GSH) or cysteine (CST) indicates that both monothiols and dithiols have the same ability in regenerating sulfhydryl (-SH) groups or chelating the metals. Inhibition of K+-p-nitrophenyl phosphatase (K+-PNPPase), the component enzyme catalyzing the K+-dependent dephosphorylation in the overall Na+-K+-ATPase reaction by these heavy metals, indicates that the mechanism of inhibition involves binding to this phosphatase. Reversal of K+-PNPPase inhibition by DTT, GSH, and CST suggests sulfhydryl groups as binding sites. Binding of 3H-ouabain, a cardiac glycocide and inhibitor of both phosphorylation and dephosphorylation, to brain fraction was significantly decreased by CH3HgCl, and this inhibition was reversed by the three thiol compounds, suggesting presence of -SH group(s) in the ouabain receptor site. Cadmium chloride failed to inhibit the binding of this receptor, indicating that the mechanics of inhibition of ATPase by CH3HgCl and CdCl2 are different from each other. The results suggest that the critical conformational property of enzyme common to both kinase (E1) and phosphatase (E2) is susceptible to CH3HgCl whereas only phosphatase is sensitive to CdCl2.