Stromal-vascular cells obtained from adult human subcutaneous adipose tissue were cultured in a chemically defined serum-free medium. In the presence of 0.2 nM triiodothyronine and 0.5 microM insulin, up to 25% of the cells were able to undergo terminal adipose differentiation within 18 d, as assessed by lipid accumulation and the expression of lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH) activities. Addition of cortisol resulted in a potent dose-dependent stimulation of the adipose differentiation process. Cortisol could be replaced by dexamethasone and partly by aldosterone, but not by sex steroids. The proportion of differentiated cells was dependent upon the age of the donor; when isolated from young adults, up to 70% of the stromal-vascular cells expressed the adipocyte phenotype as compared with 5-10% when the cells were isolated from the oldest subjects. An inverse relationship was observed between the age of the 27 normal-weight donors and the extent of GPDH expression after maintenance of the cells for 18 d in chemically defined medium supplemented with insulin, triiodothyronine, and cortisol (r = -0.787, P less than 0.001). It is concluded that adult human adipose tissue still contains precursor cells that are able to undergo adipose differentiation in vitro. This improved culture system may offer the opportunity to characterize other adipogenic factors as well as antiadipogenic factors involved in the control of adipose tissue growth.