A method is described for the isolation of a plasma membrane fraction from rat ventral prostate. This fraction is greatly enriched in (Na-", K-+)-ATPase but also contains a small amount of Mg-2+-ATPase and 5'-nucleotidase activities. 2. The activity ratio (Mg-2+ plus Na-+ plus K-+/Mg-2+ or Mg-+ plus Na-+) of the plasma membrane (Na-+, K-+)-ATPase was 1.7. The enzyme system specifically requires ATP as substrate and is inhibited by Ca-2+ or ouabain.