A p-nitrophenyl α-galactoside hydrolase is partially released when whole cells of Pseudomonas atlantica are converted to spheroplasts. The p-nitrophenyl α-glactoside hydrolase is completely inactivated by treatment of whole cells with diazonaphthalene – disulfonic acid (NDS), a reagent which does not penetrate the cytoplasmic membrane. Under the conditions used no inactivation of lactic acid dehydrogenase was observed. A specific staining procedure for this enzyme for use in electron microscopy was developed. The results with this technique in conjunction with the results of spheroplasting and NDS localization suggest that p-nitrophenyl α-galactoside hydrolase is located in or on the double-track membranes, primarily on the outer double track.