The acute in vitro and in vivo effects of long-chain fatty acids (LCFAs) on the regulation of adrenergic lipolysis were investigated in human adipose tissue. The effect of a 2 h incubation, without or with LCFA (200 mumol/l), on basal and hormonally induced lipolysis was tested in vitro on isolated fat cells. The lipolytic response to epinephrine was enhanced by suppression of the antilipolytic alpha(2)-adrenergic effect. Then, healthy lean and obese male subjects performed a 45 min exercise bout at 50% of their heart rate reserve either after an overnight fast or 3 h after a high-fat meal (HFM: 95% fat, 5% carbohydrates). Subcutaneous adipose tissue lipolysis was measured by microdialysis in the presence or absence of an alpha-antagonist (phentolamine). In vivo, a HFM increased plasma levels of nonesterified fatty acids in lean and obese subjects. In both groups, the HFM did not alter hormonal responses to exercise. Under fasting conditions, the alpha(2)-adrenergic antilipolytic effect was more pronounced in obese than in lean subjects. The HFM totally suppressed the alpha(2)-adrenergic antilipolytic effect in lean and obese subjects during exercise. LCFAs per se, in vitro as well as in vivo, suppress alpha(2)-adrenergic-mediated antilipolysis in adipose tissue. LCFA-mediated suppression of antilipolytic pathways represents another mechanism whereby a high fat content in the diet might increase adipose tissue lipolysis.