Lymphocyte-activating factor (LAF) has been shown to be produced by LPS-stimulated human adherent cells (monocytes) and peripheral leukocytes, but many non-macrophage cell lines failed to produce LAF. Other macrophage activators including latex microspheres, antigen-antibody complexes, and barium sulfate induce the production of LAF. There is a delay of 6 hr before significant amounts of LAF activity appear in the supernatant medium and maximum activity is found after 12 to 24 hr. Chromatography of concentrated crude supernatant fractions containing LAF activity on Sephadex G-100 gave two peaks of activity (approximately 85,000 and 13,000 daltons). The latter constitutes the major activity and has been purified at least 500-fold with Sephadex G-100, anion exchange, and adsorption chromatography. Optimal stimulation with LAF induces mitosis in 10% of murine thymocytes. The purified activity is sensitive to chymotrypsin and is not affected by treatment with sodium periodate, sulfhydryl reagents, and phenylmethanesulfonylfluoride. The response of thymocytes to LAF decreases with age after 10 weeks and thymocytes obtained from animals injected with cortisone or tumor-bearing animals have an increased responsiveness to LAF.