Showdomycin [2-(beta-D-ribofuranosyl)maleimide] is a nucleoside antibiotic containing a maleimide ring and which is structurally related to uridine. Showdomycin inhibited rat brain (Na+ + K+)-ATPase irreversibly by an apparently bimolecular reaction with a rate constant of about 11.01-mol- minus 1-min- minus 1. Micromolar concentrations of ATP protected against this inhibition but uridine triphosphate or uridine were much less effective. In the presence of K+, 100 MUM ATP was unable to protect against inhibition by showdomycin. These observations show that showdomycin inhibits (Na+ + K+)-ATPase by reacting with a specific chemical group or groups at the nucleotide-binding site on this enzyme. Inhibition by showdomycin appears to be more selective for this site than that due to tetrathionate or N-ethylmaleimide. Since tetrathionate is a specific reactant for sulfhydryl groups it appears likely that the reactive groups are sulfhydryl groups. The data thus show that showdomycin is a relatively selective nucleotide-site-directed inhibitor of (Na+ + K+)-ATPase and inhibiton is likely due to the reaction of showdomycin with sulfhydryl group(s) at the nucleotide-binding site on this enzyme.