ABSTRACT Mucolipidosis (ML) II and MLIII alpha/beta are two pediatric lysosomal storage disorders caused by mutations in the GNPTAB gene, which encodes an α/β‐subunit precursor protein of GlcNAc‐1‐phosphotransferase. Considerable variations in the onset and severity of the clinical phenotype in these diseases are observed. We report here on expression studies of two missense mutations c.242G>T (p.Trp81Leu) and c.2956C>T (p.Arg986Cys) and two frameshift mutations c.3503_3504delTC (p.Leu1168GlnfsX5) and c.3145insC (p.Gly1049ArgfsX16) present in severely affected MLII patients, as well as two missense mutations c.1196C>T (p.Ser399Phe) and c.3707A>T (p.Lys1236Met) reported in more mild affected individuals. We generated a novel α‐subunit‐specific monoclonal antibody, allowing the analysis of the expression, subcellular localization, and proteolytic activation of wild‐type and mutant α/β‐subunit precursor proteins by Western blotting and immunofluorescence microscopy. In general, we found that both missense and frameshift mutations that are associated with a severe clinical phenotype cause retention of the encoded protein in the endoplasmic reticulum and failure to cleave the α/β‐subunit precursor protein are associated with a severe clinical phenotype with the exception of p.Ser399Phe found in MLIII alpha/beta. Our data provide new insights into structural requirements for localization and activity of GlcNAc‐1‐phosphotransferase that may help to explain the clinical phenotype of MLII patients. The lysosomal disorder mucolipidosis II (MLII) is caused by mutations in GNPTAB encoding an α/β‐subunit precursor protein of the oligomeric Golgi‐resident GlcNAc‐1‐phosphotransferase. This enzyme forms mannose 6‐phosphate targeting signals on lysosomal enzymes prerequisite for lysosomal function. Here, we analyzed the expression of six missense and frameshift mutations identified in patients and found that they either affect ER‐Golgi transport, site‐1 protease mediated proteolytic activation or mRNA stability of the α/β‐subunit precursor protein, and associate with different clinical courses of the disease