Granulocyte colony formation by human bone marrow cells in serum-free cultures was studied using purified recombinant granulocyte colony-stimulating factor (rG-CSF). The cloning efficiency of the serum-free cultures was about 80% that of the serum-containing cultures. In addition to purified G-CSF, four ingredients were found to be essential to granulocyte colony formation: bovine serum albumin (BSA), iron-saturated human transferrin, cholesterol, and L-alpha-phosphatidylcholine. Their optimal concentrations were also investigated. Insulin was not indispensable for granulocyte colony formation, but its addition did increase the number of granulocyte colonies. Hydrocortisone was found to be inhibitory to granulocyte colony formation at high concentrations.