Cathepsin L was purified to apparent homogeneity from rat kidney. The molecular weight of the enzyme was estimated to be 30,000, but part of the enzyme was found to consist of two polypeptide chains of Mr 25,000 and 5,000. Antibody against rat kidney cathepsin L did not cross-react with rat cathepsin B or H and detected only cathepsin L in crude rat tissue preparations on immunoblotted sheets. The concentrations of cathepsin L in various rat tissues and peripheral blood cells of rats were determined by a sensitive immunoassay, in which the minimum detectable amount of cathepsin L was 20 pg/assay. The concentration of cathepsin L was found to be highest in the kidneys, where it was more than 3 times higher than in the liver, spleen, lungs, and brain. Nervous tissues, especially the cerebellar cortex, also contained fairly high concentrations of cathepsin L, but the heart, skeletal muscle, and gastrointestinal tract contained low concentrations, as did peripheral blood cells. The cathepsin L content of macrophages was 20% of that of cathepsin B. The concentrations of cathepsin L in lymphocytes, neutrophils, and erythrocytes were 10%, 20%, and less than 0.2%, respectively, of those in resident macrophages.