We have determined the three dimensional structure of the complex of human cathepsin L and E-64, an irreversible inhibitor of cysteine proteases, at 2.5 A resolution. The overall structure was similar to that of other known cysteine proteases and apparently identical to the mature region of procathepsin L. The electron density for E-64 is clearly visible except for the guanidinobutane moiety. From comparison of the active sites of cathepsin L and B, we found the following: (1) The S' subsites of cathepsin L and B are totally different because of the 'occluding loop' lying on the end of the S' subsites of cathepsin B. (2) The S2 pocket of cathepsin L is shallow and narrow compared to that of cathepsin B. (3) The S3 subsites of the two enzymes are more similar than the other subsites, but cathepsin L may accommodate a more bulky group at this site. Knowledge of the active site structure of cathepsin L should be helpful for the structure-based design of potent and specific inhibitors which are of therapeutic importance.