Human mucopolysaccharidosis VII (MPS VII, Sly syndrome) results from a deficiency of beta-glucuronidase (GUS) and has been associated with a wide range in severity of clinical manifestations. To study missense mutant models of murine MPS VII with phenotypes of varying severity, we used targeted mutagenesis to produce E536A and E536Q, corresponding to active-site nucleophile replacements E540A and E540Q in human GUS, and L175F, corresponding to the most common human mutation, L176F. The E536A mouse had no GUS activity in any tissue and displayed a severe phenotype like that of the originally described MPS VII mice carrying a deletion mutation (gus(mps/mps)). E536Q and L175F mice had low levels of residual activity and milder phenotypes. All three mutant MPS models showed progressive lysosomal storage in many tissues but had different rates of accumulation. The amount of urinary glycosaminoglycan excretion paralleled the clinical severity, with urinary glycosaminoglycans remarkably higher in E536A mice than in E536Q or L175F mice. Molecular analysis showed that the Gus mRNA levels were quantitatively similar in the three mutant mouse strains and normal mice. These mouse models, which mimic different clinical phenotypes of human MPS VII, should be useful in studying pathogenesis and also provide useful models for studying enzyme replacement therapy and targeted correction of missense mutations.