We have examined extracts of fibroblasts from patients with mannosidosis, mucolipidosis (ML) II, ML III, and normal controls for α-D-mannosidase activity against 4-methylumbelliferyl-α-D-mannopyranoside to test for the presence of the “intermediate” pH S.5 enzyme activity that has been called the “Golgi mannosidase.” Fibroblast extracts were prepared by sonication and sedimented to separate membrane-associated activities from cytosolic and lysosomal α-D-mannosidases. Membranes were extracted by salt washes (0.4 M NaCl) to desorb the lysosomal enzymes that sertimented with membranes. The α-D-mannosidase activity remaining with membranes showed many properties described for the Golgi mannosidase including: 1) an “intermediate” pH optimum (pH 5.5–6.0), 2) activity with the synthetic 4-methylumbelliferyl substrate, 3) lack of inhibitability by 200 mM methyl- α-D-mannopyranoside, and 4) partial resistance to solubilization by salt washing, and to a single extraction with the non-ionic detergent Triton X-100.This intermediate activity was the major α-D-mannosidase activity remaining in membranes from normal fibroblasts following sedimentation and salt washing, and was the major component of the α-D-mannosidase activity in extracts of fibroblasts from patients with mannosidosis, ML II, and ML III, in all of which, the lysosomal “acid” α-D-mannosidase was greatly reduced.The specific activity of intermediate α-D-mannosidase in membranes from fibroblasts from patients with mannosidosis, ML II, and ML III was not reduced compared to that of fibroblasts from controls. These studies provided no evidence to support the prior suggestion of a deficiency of “Golgi mannosidase” in ML II or ML III.