A factor that is released into the culture medium of mature adipocytes and promotes the differentiation (adipogenic conversion) of preadipocytes has been partially characterized. The factor acts in a dose-dependent manner on preadipocytes to produce up to a four-fold increase in triacylglycerol (triglyceride) content and a nine-fold increase in glycerol-3-phosphate dehydrogenase (GPDH) activity, a marker of the late phase of differentiation of preadipocytes. The material appears to be a protein, since it has a molecular weight (Superose-12 gel exclusion chromatography) of about 53kDa, an isoelectric point (pl) of 4.7–4.9, and is inactivated by the proteases papain and chymotrypsin and extremes of pH (2 and 12). Considerations of molecular weight, isoelectric point, stability to specific proteases, and especially to the action of chemical agents [the adipogenic activity is not affected by either an oxidizing (KIO4) or a reducing agent (DTT)], lead to the conclusion that the differentiation factor is distinct from known cytokines. The authors suggest that the protein be designated adipocyte differentiation factor (ADF). ADF in vivo may act as a cytokine paracrine agent to regulate the differentiation of preadipocytes.