Oligosaccharides containing terminal non-reducing alpha(1 leads to 2)-, alpha(1 leads to 3)-, and alpha(1 leads to 6)-linked mannose residues, isolated from human and bovine mannosidosis urines were used as substrates to test the specificities of acidic alpha-mannosidases isolated from human and bovine liver. The enzymes released all the alpha-linked mannose residues from each oligosaccharide and were most effective on the smallest substrate. Enzyme A in each case was less active on the oligosaccharides than alpha-mannosidase B2, even though the apparent Km value for the substrates was the same with each enzyme. The human acidic alpha-mannosidases were also found to be more active on substrates isolated from human rather than bovine mannosidosis urine. Human alpha-mannosidase C, which has a neutral pH optimum when assayed with a synthetic substrate, did not hydrolyse any of the oligosaccharides at neutral pH, but was found to be active at an acidic pH.