The intramembranous disposition of arylsulfatase C [EC 3.1.6.1] was studied. The lack of stimulation by Triton X-100 of microsomal arylsulfatase C activity indicated the outside location of the active site of the enzyme in microsomal vesicles. The exposure of arylsulfatase C on the surface of microsomal vesicles was also suggested by the binding of antibodies against the purified enzyme to intact microsomes. However, larger amounts of the antibodies were bound to microsomes in the presence of a low concentration of Triton X-100, suggesting the presence of other antigenic sites of the enzyme not available to the antibodies in intact microsomes. The treatment of solubilized and microsome-bound arylsulfatase C with transglutaminase indicated two susceptible glutamine residues per subunit of the enzyme molecule. One of the glutamine residues was labeled with transglutaminase in intact microsomes, whereas the other one became available to transglutaminase only after the addition of Triton X-100 to microsomes. These observations suggested that endoglycosidase H-sensitive carbohydrate chains of arylsulfatase C are located in the lumen of microsomal vesicles. We conclude that microsomal arylsulfatase C is a transmembranous protein and exposed on both outer and inner surfaces of the membrane.