An acylpeptide hydrolase has been purified from bovine lens tissue by anion-exchange and hydrophobic-interaction chromatography. The enzyme, purified over 27000-fold with 44% recovery, has a molecular mass of 300 kDa under native conditions. Under denaturing conditions it shows a subunit molecular mass of 75 kDa. The enzyme is inhibited by diisopropylfluorophosphate (iPr2P-F), phenylmethylsulfonyl fluoride and N-ethylmaleimide, indicating the presence of an essential serine residue and -SH group. Each subunit of the enzyme has one active serine residue which can be labelled with [3H]iPr2P-F. N alpha-blocked amino acids in L form act as competitive inhibitors of the enzyme. The antibiotics penicillin-G and ampicillin partially inhibit the enzyme. Exposure of the purified enzyme to the proteases trypsin, chymotrypsin or elastase do not result in any loss of activity. Digestion of the native enzyme with bovine trypsin generates a 55-kDa protein containing the active-site serine and a 22-kDa polypeptide, indicating the presence of a unique trypsin site. N-terminal amino acid sequencing of the 55-kDa polypeptide shows that the bovine lens enzyme has a sequence at the trypsin cleavage site identical to the porcine liver acylpeptide hydrolase sequence 196-215. The data show that the split enzyme is as active as the native enzyme towards the synthetic substrate Ac-Ala-p-nitroanilide. The enzyme activity decreases with increasing urea, but 15% of the activity remains even in the presence of 6.0 M urea. On removal of urea, complete recovery of the enzyme activity is observed. However, treatment with 1 M guanidine/HCl completely inactivates the enzyme.