We have separated four cathepsins (B, L, N and S) from rabbit spleen. They are all collagen-degrading cysteine proteinases, with Mr values of 25,250, 23,500, 34,000 and 30,000 for cathepsin B, L, N and S respectively. Cathepsins B, N and S have isoelectric points of 5.4, 6.2 and 6.8 respectively, whereas cathepsin L exhibited multiple charge forms in the range 5.0-5.7. A comparison of their specific activity against a variety of protein and synthetic substrates shows many differences. These differences can be visually illustrated through isoelectric focusing and detection of enzymic activity with protein and synthetic-substrate overlays. By using an enzyme-linked immunosorbent assay based on the binding to chicken cystatin and detection with polyclonal and monoclonal antibodies to native cathepsins B and L, no cross-reactivity of the four native enzymes was observed. Studies on the co-operative or synergistic effect in degrading collagen indicated that, of the different combinations tested, only the combination of cathepsin B and N exhibited enhanced collagenolysis.