P-aminohippurate (PAH) was evaluated as a potential blood-flow marker in steers for use in trans-organ balance studies. Because of problems in methods available for PAH determination, a high pressure liquid chromatography (HPLC) procedure was developed for the quantitation of PAH and its metabolite, N-acetyl-p-aminohippurate (aPAH). Experiments using a continuous infusion of PAH followed by a single injection of (2-glycyl-('3)H)-PAH were conducted to evaluate the distribution and kinetics of PAH in vivo;The HPLC analytic system developed for PAH and aPAH consists of a reverse-phase C-18 column with a mobile phase of 10 mM NaH(,2)PO(,4) buffer, pH 3.5, containing 30% methanol delivered at 2.5 ml/min. Absorbance at 270 nm was used for detection. Baseline separation of PAH, aPAH, and p-aminobenzoic acid (PAB) was achieved in approximately 3 minutes with this system. A linear detection response was observed for all three compounds over a concentration range of 0.12 to 64 (mu)moles/ml in aqueous standards;Six Holstein steers were used to conduct experiments evaluating the kinetics of ('3)H-PAH in vivo. The disappearance of ('3)H-PAH from blood followed a pattern described by the sum of two exponential functions. The rapid and slow components averaged t(, 1/2) of 19.5 min;PAH was distributed in a space 1.4 times that of blood volume determined with Evans blue. This is suggestive that PAH was not confined to the blood, but rather is freely diffusable into some of the extracellular space.