T he genetic relationships between the multiple variants of mucolipidosis II (I-cell disease) and mucolipidosis III (pseudo-Hurler polydystrophy) were investigated with a sensitive genetic complementation analysis procedure. These clinically distinct disorders have defects in the synthesis of a recognition marker necessary for the intracellular transport of acid hydrolases into lysosomes. Both disorders are associated with an inherited deficiency of a uridine diphosphate-N-acetyl-glucosamine: lysosomal enzyme precursor N-acetyl-glucosamine-phosphate transferase activity. We had previously shown that both disorders are genetically heterogeneous. Complementation analysis between mucolipidosis II and III fibroblasts indicated an identity of mucolipidosis II with one of the three mucolipidosis III complementation groups (ML IIIA), suggesting a close genetic relationship between these groups. The presence of several instances of complementation within this group suggested an intragenic complementation mechanism. Genetic complementation in heterokaryons resulted in increases in N-acetyl-glucosamine-phosphate transferase activity, as well as in the correction of lysosomal enzyme transport. This resulted in increases in the intracellular levels of several lysosomal enzymes and in the correction of the abnormal electrophoretic mobility pattern of intracellular beta-hexosaminidase. The findings demonstrate that a high degree of genetic heterogeneity exists within these disorders. N-acetyl-glucosamine-phosphate transferase is apparently a multicomponent enzyme with a key role in the biosynthesis and targeting of lysosomal enzymes.