A colony-stimulating factor (CSF) that stimulated human and mouse bone marrow cells to proliferate in vitro and form pure granuloid colonies was purified about 4000-fold from normal human urine. Purification procedures included concentration with polyethyleneglycol, ammonium sulfate precipitation, two chromatographic separations on DEAE- cellulose columns, gel filtration, and polyacrylamide gel electrophoresis. The molecular weight of the purified factor was estimated to be about 85,000 daltons by gel filtration, and the specific activity was found to be 10(6) or 6.7 X 10(5) colonies/mg protein using mouse or human bone marrow cells, respectively. A urinary colony-inhibiting factor was separated from the CSF on the first DEAE- cellulose column. This inhibitor suppressed the formation of pure granuloid colonies of human and mouse bone marrow cells when employed in conjunction with the purified urinary CSF.