Human urinary colony-stimulating factor (CSF-HU) has been highly purified using procedures containing DEAE cellulose, phenyl Sepharose CL-4B, Sephadex G-200, hydroxylapatite, and high performance liquid chromatography. The final preparation had a specific activity of 3.3 X 10(7) U/mg protein. Although the purified CSF-HU was not active on human monocyte-depleted bone marrow cells, it stimulated human peripheral blood monocytes obtained from five healthy volunteers to produce human active granulocytic colony-stimulating factor (G-CSF), which stimulated human monocyte-depleted bone marrow cells to form granulocytic colonies. The human G-CSF-producing activity of CSF-HU was not neutralized by polymyxin B, which is known to inhibit the effect of endotoxin. Newly produced G-CSF had an approximate molecular weight of 24,000 daltons as judged by chromatography on Sephadex G-150. These results indicate that CSF-HU stimulates human monocytes to produce human G-CSF in vitro.