Stimulation of lipolysis or adenylate cyclase activity by either isoprenaline or a tumor-derived lipid-mobilizing factor was effectively attenuated in isolated white adipocytes or in adipocyte plasma membranes pretreated with eicosapentaenoic acid (EPA) dissolved in ethanol or from mice dosed p.o. with EPA (1.25 g/kg). A similar effect was observed with docosahexanoic acid (DHA) that may be partly due to retroconversion to EPA. Stimulation of adenylate cyclase activity by forskolin, which acts directly on the enzyme without the involvement of a receptor, was also decreased in membranes of mice treated with EPA, suggesting a direct interaction between EPA and adenylate cyclase. Pertussis toxin eliminated the inhibition of lipolysis and the stimulation of adenylate cyclase by isoprenaline and lipid-mobilizing factor in the presence of EPA, but not DHA. This suggests that the attenuation of hormonal stimulation of adenylate cyclase by EPA was due, at least in part, to an inhibitory guanine nucleotide-binding protein-mediated inhibition of adenylate cyclase activity. The effect of DHA may be due to a direct inhibition of the cyclase catalytic component. The ability of EPA to preserve fat stores during the process of cachexia seems to arise from the attenuation of the stimulation of adenylate cyclase.