Para-aminohippuric acid (PAH), an indicator of renal plasma flow, is a commonly used marker of organic anion transport by the kidney. An analytical method for PAH using HPLC was developed. The method is simple, fast and requires a minimum amount of organic solvent. Sample preparation involved protein precipitation with zinc sulfate. Para-amino benzoic acid was utilized as an internal standard (IS). Chromatography was performed using a reversed-phase phenyl column with UV detection at a wavelength of 254 nm. Mobile phase consisted of 0.1 M acetic acid and acetonitrile (99:1) at a flow rate of 1 ml/min. The assay was validated over a standard concentration range from 1 to 25 microg/ml. Accuracy, precision, reproducibility and specificity of the method was established with coefficients of variation <10%. The method was sensitive and showed linear response in peak height ratio (analyte:IS) over the concentration range studied (r(2)>0.99). The assay was used to study the effect of aging on PAH excretion in the isolated perfused rat kidney model. Experiments were conducted in kidneys from young (2-3 months, n=6), adult (6-9 months, n=5) and aged (12-16 months, n=3) male Sprague-Dawley rats at an initial drug concentration of 20 microg/ml. Significant differences in kidney function (e.g. glomerular filtration rate and glucose reabsorption) were observed in aged kidneys. Despite a 5-fold reduction in glomerular filtration rate, PAH renal clearance (kidney weight-corrected) decreased by only 2-fold in aged (2.2+/-0.42 ml/min per gram) compared to young (4.6+/-0.70 ml/min per gram, P<0.05) rats. Furthermore, renal excretion ratio was significantly higher in aged rats (27+/-8.0 vs. 15+/-5.0, P<0.05). These preliminary findings challenge the 'Whole Nephron Hypothesis' that assumes parallel reductions in renal filtration and secretory capacity secondary to disease or aging.