A 1670-bp cDNA coding for a novel human cysteine protease has been isolated from a monocyte-derived macrophage cDNA library. This cDNA predicts a 329-amino acid preprocathepsin with more than 50% identity to both human cathepsin S and cathepsin L and 94% identity to a rabbit cDNA, termed OC2, recently isolated from osteoclasts. Based on its high homology to OC2, we have named the human enzyme cathepsin O. Cathepsin O mRNA was identified as a single approximately 1.7 kb transcript in cultures of 15-day-old monocyte-derived macrophages, but was not expressed in human monocytes or alveolar macrophages. When transfected into COS-7 cells, cathepsin O displayed potent endoprotease activity against fibrinogen at acid pH. This novel endoprotease may play an important role in extracellular matrix degradation.