A reliable method for obtaining osteoclast activating factor (OAF) in the culture medium of phytohemagglutinin-activated peripheral blood leukocytes is described. OAF was detected by its ability to stimulate resorption of fetal rat bone in organ culture. Most bone resorbing activity was released by activated leukocytes during the first 24 hours of culture, well before -3H-thymidine incorporation was increased. Cultures maintained for longer than 3 days showed a decrease in OAF activity. Stimulated leukocytes cultured in medium with as little as 0.01 per cent plasma showed increased -3H-thymidine incorporation and released as much OAF as leukocytes cultured with 20 per cent plasma. OAF release was stimulated by pokeweed mitogen and concanavalin A as well as by phytohemagglutinin.