Bovine kidney lysosomal α-mannosidase was purified to homogeneity and the gene was cloned. The gene was organized in 24 exons that spanned 16kb and its corresponding cDNA contained an open reading frame of 2997 bp beginning from a putative ATG start codon. The deduced amino acid sequence contained a signal peptide of 50 amino acids adjactent to a protein sequence of 949 amino acids that was cleaved into five peptides in the mature enzyme; starting with the peptide derived from the N-terminal part of this precursor, their molecular masses were 35/38 (peptide a), 11/13 (peptide b), 22 (peptide c), 38 (peptide d) and 13/15 kDa (peptide e). Variation in the degree of N-glycosylation accounts for molecular mass heterogeneities of peptides a, b and e. Peptides a, b and c were disulphide-linked. A T961°C transition, resulting in Phe321→Leu substitution, was identified in the cDNA of α-mannosidosis-affected Angus cattle. In affected Galloway cattle, a G662→A transition that causes Arg221→His substitution was identified. Phe321 and Arg221 are conserved among the α-mannosidase class-2 family, indicating that the substitutions resulted from disease-causing mutations in these breeds.