A completely serum-free culture system for L-CFU assay in vitro on 5 cases of acute non-lymphocytic leukemia (ANLL) is reported. Several agents were tested for the requirement of L-CFU assay in order to substitute the animal or human serum in the conventional culture medium. The results showed that fraction V of bovine serum albumin (2 x 10(-4) mol/L), iron saturated human transferrin (400 micrograms/ml), insulin (1 U/ml), and cholesterol (20 micrograms/ml) were indispensible components for the growth of ANLL L-CFU. Colonies of larger size could be obtained by adding 2-mercaptoethanol. HTB9-CM as the source of growth stimulating factor was used in all experiments. This method is useful for the study of L-CFU proliferation regulated by the human hemopoietic growth factors and the standardization of L-CFU assay.