1. The mode of action of acipimox (5-methyl-pyrazine carboxylic acid 4-oxide), an hypotriglyceridaemic agent, was examined in human adipose tissue and intestinal mucosa. 2. The rates of release of fatty acids and glycerol from human adipose tissue were measured in vitro. The release of fatty acids and glycerol from adipose tissue maximally stimulated by isoprenaline (10 −5 mol/l) fell by 40 and 25% respectively ( P <0.025 and P <0.025) in the presence of acipimox (10 −5 mol/l). In submaximally stimulated adipose tissue (isoprenaline 10 −7 mol/l) acipimox (10 −4 mol/l) fully inhibited release of fatty acids ( P <0.05) and glycerol ( P <0.025) to basal rates. In unstimulated adipose tissue acipimox (10 −3 mol/l) reduced the rate of glycerol release ( P <0.05), but not the rate of fatty acid release. 3. Cholesterol synthesis in jejunal mucosa was measured in vitro by the incorporation of [2- 14 C]-acetate into sterols. Addition of cholesterol to the incubation reduced [2- 14 C]acetate incorporation into sterols from 8.7 ± 2.1 (mean ± standard error) to 3.7 ± 1.0 pmol h −1 mg −1 of tissue ( P <0.01). Acipimox at 10 −4 -10 −2 mmol/l had no consistent effect on cholesterol synthesis. 4. Acipimox appears to exert its main hypolipidaemic effect by reducing lipolysis and free fatty acid flux to the liver, thereby reducing the precursor pool size of very low density lipoprotein (VLDL)-triglyceride and VLDL synthesis.