Rat tibiae and also biopsy plugs from frontal and meta carpal bones of horses were shipped in dry ice (-79[degree]C) and subsequently fixed in cold acetone (4-10[degree]C) for 12-15 hr. They were then transferred through 80%, 70% and 50% cold ethanol to a 10% aqueous solution of ethylene-diaminetetraacetic acid, sodium salt, (Sequestrene-Na2 Geigy) at 5[degree]C. The solution was constantly agitated with a magnetic stirrer and changed every 2 days for a total of 6 days. This part of the technique was quite similar to that already described by Balogh for the detection of oxidative enzymes. The tissues were than washed for a minimum of 30 min in running water at less than 10[degree]C. Frozen sections about 20 [mu]. thick were then obtained with a CO2 microtome. These were mounted on exposed and processed photographic plates (Spectroscopic Plates, type V-O, Eastman Kodak Co.). After 2-4 hr incubation at 37[degree]C in a phosphate buffer at pH 7.6, results comparable to those previosuly reported with undemineralized sections were obtained. Protease activity was localized mainly over the large osteocytes and was predominant in the mid-diaphysis. No protease activity was detected over cartilage.