Docosahexaenoic acid (DHA, C22:6), a (n-3) fatty acid in fish oil, has been shown to decrease body fat and fat accumulation in rodents. We investigated the direct effect of DHA on cell growth, differentiation, apoptosis, and lipolysis using 3T3-L1 adipocytes. Cells were treated with 25-200 mumol/L DHA containing 0.2 mmol/L alpha-tocopherol or bovine serum albumin vehicle as a control. Proliferation of preconfluent preadipocytes was not affected by the DHA treatment. When added to postconfluent preadipocytes, all concentrations of DHA inhibited differentiation-associated mitotic clonal expansion (P < 0.01). Postconfluent preadipocytes demonstrated apoptosis after 48 h with 100 mumol/L DHA and after 24 and 48 h with 200 mumol/L DHA (P < 0.01). Differentiation was examined by Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity after DHA treatment for 6 d. DHA decreased mean droplet size and percent lipid area in a dose-dependent manner (P < 0.01). GPDH activity was also decreased by DHA treatment (P < 0.01). In fully differentiated adipocytes, DHA increased basal lipolysis compared with the control (P < 0.01). These results demonstrate that DHA may exert its antiobesity effect by inhibiting differentiation to adipocytes, inducing apoptosis in postconfluent preadipocytes and promoting lipolysis.