SummaryProlidase activity from cytoplasm ofLactococcus lactissubsp.cremoris (Streptococcus cremoris)AM2 was partially purified. The enzyme hadM_r42000 and optimum activity between pH 7·35 and 8·25 in citrate, phosphate and borate buffers while in a universal buffer system an optimum pH between 8·3 and 9·0 was observed. The activity was strongly inhibited by the chelating agents E.DTA, 1,10-phenanthroline and 8-hydroxyquinoline. Inhibition was also noted with dithio-threitol,N-ethylmaleimide and bacitracin. The enzyme was active on all amino-acylproline substrates tested except Gly-Pro and Gip-Pro and also showed activity against Pro-Pro. While most prolyl amino acids tested were not hydrolysed, hydrolysis was noted with Pro-Ala and Pro-Val.K_mvalues of 20 mM and 10 mM were obtained with Phe-Pro and Met-Pro respectively; however, substrate inhibition was observed with Ile-Pro and Leu-Pro.