Plasma fatty acids from renal and hepatic veins, and arterialized hand vein obtained in 20 subjects before and after insulin infusion were separated by reversed-phase high-performance liquid chromatography following phenacyl esterification. Separation and quantification over the range 1.0-100 nmol per injection of nine fatty acids was achieved within 60 min using [2H31]palmitic acid as internal standard. Analytical recoveries were greater than 90% and the intra- and inter-assay coefficients of variation were less than 2.5 and 4.0%, respectively. Following insulin infusion, net splanchnic uptake of total fatty acids decreased from 3.0+/-0.3 to 1.0+/-0.1 micromol/kg min (p<0.01), whereas net renal balance remained neutral (-0.04+/-0.04 vs. -0.06+/-0.03 micromol/kg min, p=N.S.). Individual fatty acid balance varied from a low of 0.012+/-0.005 (myristic acid) to a high of 0.95+/-0.08 (oleic acid) micromol/kg min across the splanchnic tissues and from 0.005+/-0.002 (stearic acid) to 0.21+/-0.1 (oleic acid) micromol/kg min across the kidney. There is a substantial diversity in changes in plasma concentration and regional balance of individual fatty acid during short-term fasting and hyperinsulinemia. This method is simple, accurate, and can be applied to assess individual fatty acid metabolism in vivo.