Dogs were chronically irradiated (10 R/day; 0–400 R) or acutely irradiated (20 R/min; 0–400 R) with 60Co γ-rays. Sera were collected and assayed for colony-enhancing activity by a double-layer agar cloning technique. When test sera alone were incorporated in the feeder layers, no colony-forming units-granulocyte/macrophage formation occurred; the addition of peripheral blood leukocytes to such test sera-containing feeder layers resulted in formation of colonies, the number of which was directly related to the cumulative radiation dose. Serum-enhancing factor activity indirectly increased colony formation by stimulating feeder layer leukocytes to generate increased levels of colony-stimulating activity. Levels of serum-enhancing activity were higher at similar dose levels following acute gamma irradiation then following chronic irradiation.