The adipose conversion of 3T3-L1 fibroblasts depends on serum factors, one of which has been identified as GH. Human serum extracts obtained by heat treatment do not contain intact GH, as shown by RIA, but still support adipose conversion. After organic solvent extraction followed by reversed phase HPLC, two adipogenic fractions can be purified. Their identification as cortisol and cortisone follows from the observations that both glucocorticoid hormones are strongly adipogenic in physiological concentrations and elute upon reversed phase HPLC with retention times identical to those of the adipogenic fractions. After derivatization by acetylation, the adipogenic fractions purified from human serum again do not differ from C-21 monoacetyl cortisol and -cortisone with regard to adipogenic activity and behavior on reversed phase HPLC and TLC. Treatment of heat extracts from human serum with cortisol antibodies strongly reduced their adipogenic activity which could be restored only after addition of cortisol to the original concentration. We conclude from these results, that the adipogenic activity of human serum depends mainly on its glucocorticoid (as well as GH) concentration.