A highly purified (Na⁺, K⁺)-ATPase from the rectal gland of Squalus acanthias and from the electric organ of Electrophorus electricus has been used to raise antibodies in rabbits. The 97,000 dalton catalytic subunit and glycoprotein derived from the rectal gland of spiny shark were also used as antigens. The two (Na⁺, K⁺)-ATPase holoenzymes and the two shark subunits were antigenic. In Ouchterlony double diffusion experiments, these antibodies formed precipitation bands with their antigens. Antibodies prepared against the two subunits of shark holoenzyme also formed precipitation bands with their antigens and shark holoenzyme, but not with eel holoenzyme. These observations are in good agreement with inhibitory effect of these antibodies on the catalytic activity of (Na⁺, K⁺)-ATPase both from the shark and the eel, since there is very little cross-reaction between the shark anticatalytic subunit antibodies and the eel holoenzyme. The maximum antibodies titer of the anticatalytic subunit antibodies is found to be 6 weeks after the initial single exposure to this antigen. Multiple injections of the antigen increased the antibody titer. However, the time required to produce the maximum antibody titer was approximately the same. These antibodies also inhibit catalytic activity of (Na⁺, K⁺)-ATPase vesicles reconstituted by a slow dialysis of cholate after solubilization of the enzyme in a presonicated mixture of cholate and phospholipid. In these reconstituted (Na⁺, K⁺)-ATPase vesicles, effects of these antibodies on the fluxes of Na⁺, Rb^+, and K⁺ were investigated. Control or preimmune serum had no effect on the influx of ²²Na⁺ or the efflux of ⁸⁶Rb⁺. Immunized sera against the shark (Na⁺, K⁺)-ATPase holoenzyme, its glycoprotein or catalytic subunit did inhibit the influx of ²²Na⁺ and the efflux of ⁸⁶Rb⁺. It was also demonstrated that these antibodies inhibit the coupled counter-transport of Na⁺ and K⁺ as studied by means of dual labeling experiments. However, this inhibitory effect of the antibodies on transport of ions in the (Na⁺, K⁺)-ATPase vesicles is manifested only on the portion of energy and temperature dependent alkali metal fluxes, not on the portion of ATP and ouabain insensitive ion movement. Simultaneous determination of effects of the antibodies on ion fluxes and vesicular catalytic activity indicates that an inhibition of active ion transport in reconstituted (Na⁺, K⁺)-ATPase vesicles appears to be due to the inhibitory action of the antibodies on the enzymatic activity of (Na⁺, K⁺)-ATPase molecules incorporated in the vesicles. These findings that the inhibitory effects of the antibodies specific to (Na⁺, K⁺)-ATPase or to its subunits on ATP and temperature sensitive monovalent cation transport in parallel with the inhibitory effect of vesicular catalytic activity by these antibodies provide direct evidence that (Na⁺, K⁺)-ATPase is the molecular machinery of active cation transport in this reconstituted (Na⁺, K⁺)-ATPase vesicular system.