The effect of a variety of cytokines on lipid metabolism in 3T3 L1 mouse fibroblasts and adipocytes was studied. Uptake of [3H]acetate by adipocytes and heparin-releasable lipoprotein lipase activity was inhibited after treatments of the cells with picomolar concentrations of recombinant human tumor necrosis factor alpha (rHuTNF-alpha), human tumor necrosis factor beta (rHuTNF-beta, also called lymphotoxin), murine interferon-gamma (rMuIFN-gamma), and a human hybrid interferon-alpha [rHuIFN-alpha 2/alpha 1 (Bgl II)]. Recombinant human interferon-gamma (rHuIFN-gamma), natural human colony-stimulating factor (HuCSF), and human interleukin 2 (HuIL-2) had no effect. Similar though less-marked suppression of [3H]acetate uptake by cytokines was seen in 3T3 L1 fibroblasts. Cytokines inhibited the incorporation of [3H]acetate into both membrane and storage lipids in the adipocytes. In addition to blocking lipid uptake and synthesis, rHuTNF-alpha and -beta, and rMuIFN-gamma stimulated the release of free fatty acid into the medium from adipocytes. Binding studies suggest that rHuTNF-alpha and rHuTNF-beta compete for the same cell-surface receptor on 3T3 L1 adipocytes, while rMuIFN-gamma binds to a separate receptor. The binding of rTNF-alpha to both adipocytes and fibroblasts can be significantly enhanced by preexposure of the cells to rMuIFN-gamma. There appear to be both high- and low-affinity receptors for rHuTNF-alpha on adipocytes, whereas fibroblasts exhibit a single class of high-affinity receptors. These results suggest that a variety of structurally distinct cytokines possess lipid mobilization activity, which may be of critical importance to the host in defense against infection or malignancy.