A rapid and efficient isocratic high-performance liquid chromatographic method for studying the metabolism of phosphoenolpyruvate and uridine diphosphate glucose (UDPG) has been developed. For each compound this method can measure tissue concentrations in the range 0.1-1000 nmol/g of tissue and determine specific radioactivity. All measurements can be performed in 200 mg of tissue. The recoveries of uridine diphosphate [6-3H]glucose and phosphoenol[1-14C]pyruvate from liver tissue homogenates were 97 and 99%, respectively. Following intra-arterial infusion of [6-3H]glucose and [U-14C]lactate in conscious rat, the concentration and specific radioactivity of phosphoenolpyruvate and UDPG were determined in rat liver. The method may be applied to experimentation in small animals using radiolabelled precursors in order to quantitate in vivo the glycogenic and gluconeogenic fluxes.