Normal N-acetylglucosamine 1-phosphotransferase activity toward mono- and oligosaccharide acceptor substrates was detected in cultured skin fibroblasts from mucolipidoses II and III patients who were designated as variants (one of four mucolipidosis II and three out of six mucolipidosis III patients examined). The activity toward natural lysosomal protein acceptors was absent or deficient in cell preparations from all patients with classical as well as variant forms of mucolipidoses II and III. Complementation analysis, using fused and cocultivated mutant fibroblast combinations, revealed that, while cell lines with variant mucolipidosis III constituted a complementation group distinct from that of classical forms of mucolipidoses II and III, the variant mucolipidosis II cell line belonged to the same complementation group as did the classical forms. In contrast to the mutant enzyme from variant mucolipidosis III patients that failed to recognize lysosomal proteins as the specific acceptor substrates, the activity toward alpha-methylmannoside in the variant mucolipidosis II patient could be inhibited by exogenous lysosomal enzyme preparations (bovine beta-glucuronidase and human hexosaminidase A). These findings suggest that N-acetylglucosamine 1-phosphotransferase is composed of at least two distinct polypeptides: (1) a recognition subunit that is defective in the mucolipidosis III variants and (2) a catalytic subunit that is deficient or altered in the classical forms of mucolipidoses II and III as well as in the mucolipidosis II variant.