Supernatants of phytohemagglutinin-stimulated human tonsil cells contain two growth inhibitory factors. These factors, called inhibitors of DNA synthesis (IDS), reduce (3)H-thymidine incorporation into mitogen-stimulated lymphocytes and into growing HeLa cells. By Sephadex chromatography, these factors have volumes of distribution corresponding to about 80,000 and 40,000 daltons. Both factors inhibit the activity of calf thymus DNA polymerase alpha in cell-free assays (termed inhibitor of DNA polymerase, IDP). The larger factor, which is chromatographically separable from alpha-lymphotoxin (alpha-LT), is completely inactivated by heating at 70 degrees C for 15 min. This treatment does not destroy alpha-LT. Using supernatants from PHA-stimulated tonsil cells cultured for 5 days in serum-free medium, we attained a 150-fold purification with a succession of molecular sieving, ion exchange, and adsorption chromatographic procedures. Although not purified to homogeneity, the extensive copurification of IDS and IDP activities and their identical heat inactivation profiles suggest that they are the same entity. IDP separated free of alpha-LT inhibits thymidine incorporation into HeLa cells without causing cell death. alpha-LT purified free of IDS does not inhibit thymidine incorporation into HeLa cells, not even at concentrations 7000 times that necessary to kill 50% of growth-inhibited L cell cultures.