Fibroblasts from patients with Morquio B syndrome contain normal numbers of 0-galactosidase molecules with normal turnover but strongly reduced activity per enzyme molecule. Various substrate affinities are abnormal: the K, for methylum belliferyl (MU)-P-galactoside is 4-10-fold elevated and affinity for keratan sulphate and oligosaccharides, isolated from Morquio B urine, was not detectable. In contrast, these substrate affinities are normal for 0-galactosidase in adult type GM 1 -gangliosidosis fibroblasts. Cell hybridization studies demonstrate that Morquio B syndrome and infantile and adult type GMI-gangliosidosis belong to the same complementation group. From these results we conclude that Morquio B syndrome is caused by a mutation in the structural gene for P-galactosidase, which is allelic to the mutations in infantile and adult type GMI-gangliosidosis. Urinary excretion of keratan sulphate and oligosaccharides is abnormal in Morquio B syndrome but normal in adult type GM, -gangliosidosis. The catalytic properties of 6-galactosidase in Morquio B syndrome and GM I-gangliosidosis provide a possible explanation for the distinct clinical manifestations in these disorders.