Spleen cells from fibrosarcoma-bearing mice elaborated a factor of 30 to 50,000 daltons in m.w. that was capable of inhibiting DNA synthesis and DNA polymerase activities of mitogen-stimulated lymphocytes plus unrelated cell types. The cells responsible for producing these factors were characterized as anti-Thy 1 serum- and hydrocortisone-sensitive mildly nylon wool adherent spleen cells (suggesting a suppressor T cell). Both DNA polymerases alpha (α) and beta (β) were significantly inhibited by prepared supernatants from cultures of tumor-bearing host (TBH) spleen cells. DNA polymerase polymerase (IDP). Basal levels of TBH lymphocyte DNA synthesis and polymerase α activity were always higher than their normal counterparts; however, upon mitogen stimulation this was reversed. Normal animal spleen cell DNA polymerase α activity exhibited a direct correlation with mitogen-induced DNA synthesis. In the presence of mitogen, normal spleen cells demonstrated a significant increase in DNA synthesis with a corresponding increase in DNA polymerase α activity, whereas TBH spleen cells upon mitogen stimulation resulted in a concomitant decrease in DNA synthesis and polymerase activity. Inhibition kinetics revealed that IDP was not a competitive inhibitor of DNA polymerase α and did not bind directly to the DNA template in the absence of polymerase. The kinetics imply the production of an enzyme-substrate-inhibitor complex which cannot undergo further reaction to yield a normal product. The data suggest that substances elaborated by tumor-induced suppressor cells operate at a molecular site of DNA polymerases resulting in inhibited cellular proliferation.