A human colony-stimulating factor (CSF)-producing cell line, T3M-5, has been established in vitro from a squamous cell carcinoma of the thyroid gland transplanted into athymic nude mice [congenitally athymic BALB/c (nu/nu) mice; Central Institute for Experimental Animals, Kawasaki, Japan]. Contaminating fibroblasts derived from a host nude mouse were eliminated by treatment with antiserum raised against nude mouse cells. T3M-5 cells have been continuously propagated during 3 years. The cells grew in a monolayered sheet with about 22 hours of population-doubling time and showed about 40% plating efficiency. The cells exhibited an epithelium-like morphology resembling the structure of the original tumor and showed tumor takes when inoculated into nude mice. Chromosome analysis revealed the cell line to be a human aneuploid line with a hypertriploid mode. The cells possessed the characteristic function of human CSF production in vitro and produced marked neutrophilia in tumor-bearing nude mice that were inoculated with the cultured cells. The molecular weight of the CSF was estimated at about 27,000 and was stable over the pH range 1.0-9.0 at 4 degrees C for 21 hours. The CSF activity was destroyed by either trypsin or chymotrypsin, but it resisted neuraminidase, DNase, and RNase. The cells could be well propagated in roller bottles. About 100 liters of the conditioned medium was obtained with the roller bottle culture method, which formed approximately 500,000,000 colonies of human bone marrow cells. The rate of recovery of CSF activity from the gel-filtration column was high (68.9%). This cell line is therefore expected to aid in the large-scale preparation of human CSF.