Human granulocyte-macrophage colony-stimulating factor (GM-CSF) was purified from 3 liters of serum-free conditioned medium of the Hodgkin's tumor cell line L428 KSA. The conditioned medium contained a high specific activity of 2.5 X 10(5) units of total colony-stimulating factor per mg protein. Colony-stimulating factor activity was determined by colony formation by human fetal liver cells or mouse bone marrow cells. The latter bioassay discriminated colony-stimulating factor 1, a subclass specific for monocyte/macrophage production, and G-CSF, specific for granulopoiesis, from GM-CSF. The starting material contained predominantly GM-CSF with CSF-1 and G-CSF constituting 10% and 12%, respectively, of the total activity. A seven-stage purification scheme was employed. The first stage involved concentration by batch chromatography on calcium phosphate gel. Subsequent stages involved gel filtration on Ultrogel AcA44, affinity chromatography on concanavalin A-Sepharose, batch chromatography on calcium phosphate gel and high-performance liquid chromatography on C1 reversed-phase (TSK TMS-250), gel permeation and C8 reversed-phase columns. The purified material showed a single disperse band, having an Mr of 30,000, by silver staining on sodium dodecyl sulfate polyacrylamide gel electrophoresis. An amino-terminal sequence of 20 amino acids was determined in a gas-phase sequencer with 500 ng of purified material. The sequence was identical to that predicted from the cDNA sequence. It was active on human fetal liver cells with half-maximum colony formation at 1 X 10(-12) M, but was not active on mouse bone narrow cells.