A cDNA for a novel human papain-like cysteine protease, designated cathepsin F, has been cloned from a l gt10-skeletal muscle cDNA library. The nucleotide sequence encoded a polypeptide of 302 amino acids com-posed of an 88-residue propeptide and a 214-residue mature protein. Protein sequence comparisons revealed 58% homology with cathepsin W; about 42–43% with cathepsins L, K, S, H, and O; and 38% with cathepsin B. Sequence comparisons of the propeptides indicated that cathepsin F and cathepsin W may form a new cathepsin subgroup. Northern blot analysis showed high expression levels in heart, skeletal muscle, brain, testis, and ovary; moderate levels in prostate, placenta, liver, and colon; and no detectable expression in peripheral leukocytes and thymus. The precursor polypeptide of human recombinant cathepsin F, produced in Pichia pastoris , was processed to its active mature form autocatalytically or by incubation with pepsin. Mature cathepsin F was highly active with comparable specific activities toward synthetic substrates as reported for cathepsin L. The protease had a broad pH optimum between 5.2 and 6.8. Similar to cathepsin L, its pH stability at cytosolic pH (7.2) was short, with a half-life of approximately 2 min. This may suggest pH Activity Profile and pH Stability— Initial rates of substrate hy- drolysis were monitored as described above. The pH activity profile of human cathepsin F was obtained at 1 m M substrate (Z-FR-MCA) con- centration ([S] , K m , where the initial rate, v o , is directly proportional to the k cat / K m value). The following buffers were used for the pH activity profile: 100 m M sodium citrate (pH 2.8–5.6) and 100 m M sodium phosphate (pH 5.8–8.0). All buffers contained 1 m M EDTA and 0.4 M NaCl to minimize the variation in ionic strength. A three-protonation model (22) was used for least square regression analysis of the pH activity data. The data were fitted to the following equation. 100 m M sodium acetate buffer, pH 5.0 (lysosomal and in potassium phosphate buffer, 7.2 (cytosolic At appropriate time intervals, aliquots of the incubation mixture withdrawn, and the activity was measured using fluorogenic assay