Different clinical expressions of acid α-glucosidase deficiency have been described. The present study was undertaken to investigate the basic metabolic defect in the infantile and adult forms of the disease. Acid α-glucosidase (EC 126.96.36.199) was purified from normal and from adult acid α-glucosidase deficiency fibroblasts. The pH optimum; Michaelis constant; electrophoretic mobility in starch; thermal denaturation at pH 4.0 and 7.0; and inhibition by turanose, α-methylglucoside, and trehalose were the same in purified enzyme from normal and mutant cells. Placental acid α-glucosidase was purified to, or near, homogeneity. Monospecific antibodies raised against the enzyme in each of three enzyme peaks obtained from the last purification step were found to cross-react with the enzyme of all three peaks, and with purified, normal fibroblast enzyme. Cross-reacting material (CRM) also was identified in fibroblast lysates from normal subjects and from both forms of acid α-glucosidase deficiency. The amount of CRM in the adult form appeared to be significantly less than in normal cells or cells from the infantile form. Enzyme activity was demonstrated in the immune complexes of the normal and adult acid α-glucosidase deficiency fibroblasts, but not of the infantile form. Competition for antibody binding sites was observed between normal and both types of mutant enzyme. The findings indicate that this case of infantile acid α-glucosidase deficiency is the result of a structural gene mutation which causes the synthesis of a catalytically inactive (CRM-positive) enzyme protein. It appears that in the adult form, the mutation causes a reduction in the amount of the enzyme protein present in the cells.