The extracellular compartment where bone resorption occurs, between the osteoclast and bone matrix, is shown in this report to be actively acidified. The weak base acridine orange accumulates within this compartment but dissipates after incubation with ammonium chloride. Upon removal of ammonium chloride, the cells are able to rapidly reacidify this compartment. The highly convoluted plasma membrane of the osteoclast facing this acidic compartment (ruffled border) is shown to contain a 100-kD integral membrane protein otherwise present in limiting membranes of lysosomes and other related acidified organelles