To determine the tissue distribution of glycogen debranching enzyme, we used immunoblot analysis with a polyclonal antibody prepared against purified porcine muscle debranching enzyme. Debranching enzyme was identified in porcine brain, kidney, cardiac muscle, skeletal muscle, liver, and spleen; and in human liver, skeletal muscle, lymphocytes, lymphoblastoid cells, skin fibroblasts, cultured chorionic villi, and amniocytes. In each of these tissues the debranching enzyme band was 160 kd. To determine the molecular basis for glycogen storage disease type III at the protein level, tissues from 41 patients with glycogen storage disease type III were also subjected to immunoblot analysis. Three patients having isolated transferase deficiency with retention of glucosidase activity (type IIID disease) had nearly normal amounts of cross-reactive material. In the remaining patients (both transferase and glucosidase deficiency), debranching enzyme was either absent or greatly reduced. These latter patients included 31 with disease that appeared to involve both liver and muscle (type IIIA), four with disease that was present only in the liver (type IIIB), and three with unknown muscle status. In patients with both type IIIA and type IIIB disease, debranching enzyme protein was absent in skin fibroblasts, lymphoblastoid cells, and lymphocytes. The parents of two patients with type IIIA disease had an intermediate level of debranching enzyme protein, consistent with their presumed heterozygote state. An immunoblot analysis of cultured amniotic fluid cells from a woman whose fetus was at risk for type IIIA disease predicted an unaffected fetus; the prediction was confirmed postnatally. Thus Western blot analysis offers an alternate method of prenatal diagnosis for the most common form of glycogen storage disease type III.