To define the cause of clinical heterogeneity in glycogenosis type II we have studied the inheritance and molecular nature of acid alpha-glucosidase deficiency in a rare family with severe infantile as well as mild late-onset variants of this disease. The (mutant) acid alpha-glucosidase alleles of crucial family members were segregated in human-mouse somatic cell hybrids to investigate their individual function. Two types of mutant alleles were identified. The first leads to complete deficiency of acid alpha-glucosidase. Homozygosity of this allele is demonstrated in three cases of severe infantile glycogenosis type II in the family under study. The second mutant allele is characterized by a reduced net production of catalytically active acid alpha-glucosidase, resulting in partial enzyme deficiency. The eldest patient in the family, with very mild clinical symptoms, is shown to be a compound heterozygote having both types of mutant alleles. These studies emphasize the effect of allelic diversity on the level of residual acid alpha-glucosidase activity and on the clinical course of glycogenosis type II.