Spleen cells were collected from normal mice and cultured in a medium containing 20% calf serum. Addition of lipopolysaccharide (LPS) in the culture significantly increased the production of granulocyte-macrophage colony-stimulating factor (GM-CSF), and a maximum induction was attained in 5 days. Irradiation of the spleen cells with 300 to 3,000R X-rays also enhanced the production of GM-CSF, but there was a latent period of about 5 days before the factor appeared in the culture medium. The observed difference between LPS and X-rays in the timing of inducing GM-CSF production in the spleen cell culture was consistent with the difference in timing of the increase of spleen cell proliferation observed in animals after the administration of LPS or during recovery from damages by X-irradiation. It was observed furthermore that the X-ray-induced GM-CSF differed from the LPS-induced GM-CSF in its molecular properties; the X-ray-induced factor was represented by an acidic (pI = 3.0) 70,000-dalton species, while the LPS-induced factor was much smaller in size (M.W. 20,000) and less acidic (pI = 5.4). These results suggest that different mechanisms of GM-CSF production operate in the spleen in response to either LPS or X-rays.