We recently published the primary structure and inhibition data of the barley grain aspartic proteinase (HvAP, Hordeum vulgare aspartic proteinase) which revealed similarity to mammalian cathepsin D and yeast aspartic proteinase A. Here we present evidence, based on Km and kcat values for the enzyme as well as on its cleavage sites in haemoglobin, the insulin B-chain, glucagon and melittin, that the similarity extends to its hydrolytic specificity. Like the animal and microbial aspartic proteinases, HvAP preferentially cleaves peptide bonds between amino acid residues with large hydrophobic side chains. The narrow hydrolytic specificity of HvAP suggests that plant aspartic proteinases may perform regulatory functions by limited proteolysis.