The significance of neuraminidase deficiency reported to be the primary defect in mucolipidosis II has been evaluated by determination of this enzyme activity in cultured fibroblasts, culture medium, and leucocytes from homozygote and heterozygous carriers of the disease. A new and sensitive fluorometric assay of neuraminidase was used with sodium (4-methylumbeliferyl-alpha-D-N-acetylneuraminate) as substrate. We report: 1) nearly total deficiency of neuraminidase in mucolipidosis fibroblasts, 2) partial deficiency of this enzyme in leucocytes of one patient, 3) this decreased activity ceases to exist following Triton X-100 treatment, and 4) intermediary mean neuraminidase activity in fibroblasts and leucocytes from obligate heterozygotes. Although these results would be consistent with the suggestion that neuraminidase deficiency is the primary defect in this disease, evidence from the work of other authors suggests that the enzyme deficiency results from a secondary effect of the mucolipidosis II mutation.