A high-performance liquid chromatographic (HPLC) assay method has been developed for the quantitative determination of iothalamate and p-aminohippuric acid (PAH) concentrations in serum and urine samples in the male rat. Glomerular filtration rate (GFR) was measured as clearance of iothalamate, while effective renal blood flow (ERBF) was measured as clearance of PAH. The method is simple, rapid and sensitive and detects iothalamate and PAH in rat serum and urine following administration of bolus doses and continuous infusions of iothalamate and PAH. Samples of serum and urine were deproteinized with two volumes of acetonitrile containing the internal standard, and an aliquot chromatographed on a C18 reversed-phase column. The mobile phase was comprised of 0.1 M sodium phosphate with 1.2 mM tetrabutylammonium phosphate: methanol, 85:15 (v/v), at a flow rate of 1.0 mL/min. The analytical column eluate was monitored with a UV detector at 254 nm with quantitation achieved using peak-height ratios. The precision of the method was 6.6 and 3.6% for iothalamate in serum and urine, and 5.6 and 4.9% for PAH in serum and urine, respectively. The lower limit of quantitation was 0.63 microgram/mL for iothalamate and 1.25 microgram/mL for PAH in serum, and 3.1 microgram/mL for iothalamate and 1.5 microgram/mL for PAH in urine. Recovery of iothalamate from serum and urine was 99.9 and 93.5%, respectively. Recovery of PAH from serum and urine was 99.8 and 92.6%, respectively. The present study demonstrated that non-radioactive iothalamate and PAH can be measured simultaneously using a HPLC assay to measure GFR and ERBF in the male rat.