The concept of humoral control of granulopoiesis is supported by the isolation and testing of two regulatory substances, chalone and antichalone. Rytomaa proposed specific criteria for chalone: (1) inhibitor of cell mitosis; (2) tissue specific but not species specific; (3) reversible action; (4) no cytotoxicity. Antichalone reverses or negates the action of chalone. Using rat granulocytes obtained from peritoneal exudate after intraperitoneal injections of sterile polyvinylpyrrolidone (PVP), we produced "conditioned media" by incubating these cells in a balanced salt solution. The "conditioned media" was fractioned on Sephadex gel filtration. Fractions concentrated by lyophilization were then individually tested for inhibitory properties against a target tissue, in this case, rat marrow suspension. Incorporation of tritiated thymidine into cells synthesizing DNA was measured by liquid scintillation counting and autoradiography. Total cell count of the suspension, pre- and postincubation, confirmed no cell loss occurred. Specific fractions consistent in their elution pattern from gel filtration caused 40-60% decreased incorporation of tritiated thymidine into rat marrow cells as compared to their controls. Autoradiographic results correlated well with these findings. We have demonstrated that rat granulocytes produce an inhibitory substance which reduces or retards the incorporation of tritiated thymidine into DNA synthesizing cells. Application of these techniques to the separation of chalone and antichalone from the blood of normal volunteers and patients with myeloproliferative disorders will follow.