Aminoacylase I (EC 3.5.1.14) is one of the most abundant enzymes in the cortical region of mammalian kidney. Both the porcine and the human enzyme were overexpressed using baculovirus expression vector systems and purified by hydrophobic interaction chromatography and anion-exchange chromatography. The resulting preparations were analyzed for structural and functional identity with the corresponding enzymes isolated from kidney. The dansyl method as well as mass spectroscopy confirmed N-terminal blocking. For the porcine enzyme, atomic absorption spectroscopy yielded the correct metal content (one zinc per subunit). Kinetic analyses showed identical Km values for the expression products and the enzymes isolated from kidney. By contrast, the porcine enzyme when overexpressed in Escherichia coli had a much lower specific activity. Comparative substrate specificity studies with natural and recombinant human aminoacylase and 16 different N-acetyl-L-amino acids showed that, among the derivatives of proteinogenic amino acids, N-acetyl-L-methionine was the best substrate, followed by acetylated glutamate, leucine, alanine, and valine. These amino acids are also the most abundant residues at the N-termini of acetylated proteins. This suggests that kidney aminoacylase may be involved in the salvage of amino acids by hydrolyzing acetyl amino acids released from proteins.