To examine whether a human colony-stimulating factor (CSF)-producing cell line, T3M-1, can propagate and secrete CSF without protein supplements, long-term cultivation of the cells was carried out in a protein-free chemically defined medium. By the use of stepwise decreases in the fetal bovine serum concentration, continuous growth of T3M-1 cells has been established in a protein-free F-10 medium. The cells designated T3M-1-T2 have been propagated in this medium for 5 years. The population-doubling time of the cells is about 30 hr. Addition of serum stimulated the cell growth (population-doubling time, 17 hr) but did not increase the saturation density. The cells secreted large amounts of CSF (2000 colonies stimulated by 1 ml of the conditioned medium). Addition of serum to the culture increased CSF activity in the conditioned medium (3400 colonies/ml). The results showed that a human cancer cell line, T3M-1-T2, could be propagated in a protein-free chemically defined medium and secrete large amounts of CSF. The cells will serve as an excellent model for better understanding of the cell growth and production of CSF in the absence of any serum contamination.