We have developed a completely defined serum-free medium that supports the growth of Swiss 3T3-L1 fibroblasts to nearly the same extent as Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. With ASF301 medium [former name, RITC 80-7; Yamane et al. Exp. Cell Res. 134, 470 (1981)], most of the 3T3-L1 cells survived for at least 10 days, but did not grow. ASF301 medium contains insulin and mouse-epidermal growth factor as growth factors, which are termed "progression factors". So we examined the effects of "competent factors" on the proliferation of 3T3-L1 preadipocytes. Growth in the medium supplemented with competent factors reached a confluent monolayer in 6-7 days. Furthermore, it was confirmed that 3T3-L1 cells grown in the serum-free medium retained the properties of differentiation into adipocytes. Our serum-free medium should be a useful tool for research on the growth and differentiation of 3T3-L1 preadipocytes.