Cathepsin A was purified approximately 550-fold with an overall yield of 4% from bovine spleen crude extracts by successive chromatographies on DEAE-Sephadex A-50, phenyl-Toyopearl 650C, and Con A-agarose. PAGE of the purified enzyme without 2-mercaptoethanol revealed an apparent molecular size of 110 kDa, and SDS-PAGE with 2-mercaptoethanol gave two polypeptide bands corresponding to 32 and 25 kDa and without 2-mercaptoethanol a single polypeptide 52 kDa band. These results indicate that the enzyme has an (alpha beta)2 tetrameric structure in which the alpha (32 kDa) and beta (25 kDa) subunits are linked by disulfide bond(s). The enzyme exhibited peptidase activities, hydrolyzing various Z-dipeptides with optimum pHs between 5.0 and 5.8. The hydrolytic rate for Z-Phe-Ala was 15 times higher than that for Z-Glu-Tyr, the traditional cathepsin A substrate. The enzyme also catalyzed the hydrolysis of the C-terminal amino acids of RCM-RNase A and showed esterase activity toward BTEE at pH around 7.5. DFP and TPCK completely inhibited both peptidase and esterase activities, and [1,3-3H]DFP was bound to the alpha subunit. All these results support the fact that the enzyme is a serine carboxypeptidase. The N-terminal amino acid sequences of the alpha and beta subunits are highly homologous to those of the human protective protein in galactosialidosis, strongly supporting the identity between cathepsin A and the protective protein.