Four forms of mouse granulocyte/macrophage colony-stimulating factor (GM-CSF) were purified 100,000-fold from mouse lung conditioned medium. Each of the CSF species stimulated the formation of both granulocyte and macrophage colonies, and half-maximal stimulation in the semi-solid mouse bone-marrow colony assay occurred at 1 pm. The four GM-CSF species exhibited similar charge microheterogeneity, focusing between pH 4.2 and pH 5.2. On SDS/polyacrylamide gels two of the GM-CSF sub-species had apparent Mr values of 23,000, and the other two, 21, 000. Treatment with neuraminidase decreased the Mr values of these two sets to 21,000 and 19,000 respectively. Incubation with endoglucosidase F decreased the charge heterogeneity and the Mr of all species to 16,500. A gas-phase radioiodination procedure was used to incorporate 2-3 atoms of 125I/molecule into purified GM-CSF without any loss of biological activity. The 125I-labelled GM-CSF was analysed on a microbore reversed-phase h.p.l.c. column to determine its specific radioactivity directly. This 125I-labelled GM-CSF molecule is suitable for cell-surface receptor-binding studies.