Treatment of a 128 kDa mouse nardilysin with trypsin initially produced an active 105 kDa N-terminally cleaved form. Continued trypsin digestion occurred at the C-terminus, producing inactive core species of ~92, 76.5, and 62 kDa. Protease V8 digestion generated a stable ~105 kDa form, nardilysin V 8 , that was cleaved near the N-terminal trypsin site. The ~105 kDa nardilysin V 8 exhibited the same K m as did the uncleaved enzyme for substrates of the type Abz-GGFX 1 X 2 X 3 VGQ-EDDnp, where X residues were varied. However, k c a t for nardilysin V 8 was 5-6 times greater. Both uncleaved nardilysin and nardilysin V 8 are inhibited by NaCl; however, nardilysin V 8 exhibits an IC 5 0 of ~2 mM compared to an IC 5 0 of ~50 mM for uncleaved nardilysin. Nardilysin V 8 is more sensitive to inhibition by phosphate buffer. Treatment of nardilysin V 8 with trypsin generated primarily the 92 kDa form which was inactive. Attempts to express nardilysin as a 105 kDa truncated N-terminal form or as a C-terminally truncated form led to inactive proteins.