Cytosolic bovine brain pyroglutamyl aminopeptidase was purified from whole brain by chromatography with DEAE sepharose, G100 gel filtration and ATS 4B affinity chromatography. An overall recovery of 14.4% and a purifiaction factor of 247 was achieved. The relative molecular mass of PAP-I, determined by SDS PAGE, was found to be 24,680 Da. The partially purified and purified enzyme was found to be relatively unstable under assay conditions. Of all the additives tested as stabilisers only BSA was found to stabilise the enzyme, during assay and during storage. A minimum concentration of approximately 0.1% w/v BSA was found to achieve optimum stabilisation. PAP-I was confirmed to be a cysteine protease based on its sensitivity to IAA, NEM and iodoacetamide. The enzyme was also inhibited by DEPC and EDC, suggesting that His and Glu and/or Asp are also involved in events at the active site. These results were confirmed by the protective effect of the competitive inhibitor TRH on inactivation of PAP-I by these modifying reagents. Cys and His may constitute the nucleophilic and imidazole residues of the active site, while Asp or Glu might constitute the third part of the active site. Tyr, Lys, Arg and Ser are not involved in the expression of activity. Initial chemical modification studies demonstrated that EG NHS modified PAP-I showed almost a two fold increase in thermostability over the native enzyme. AA-NHS modified enzyme, did notshow any increase in thermostability.