We describe a sensitive, reproducible method for the simultaneous determination of the ATP catabolites inosine, hypoxanthine, xanthine, and uric acid in biological samples and organ perfusate using reverse-phase chromatography and multiwavelength detection at 254, 270, and 292 nm. Sample preparation includes precipitating proteins with perchloric acid, neutralizing the sample, passing the supernatant over a polyethyleneimine column, and analyzing the collected fractions by high-performance liquid chromatography. Addition of metal chelators to the perchloric acid resulted in increased values for xanthine, hypoxanthine, and uric acid. The method was sensitive (limit of detection, 0.08 nmol on column; S/N = 4) and linear over the range 0.5-30 microM. Precision and accuracy of the method were evaluated for lung tissue and lung perfusate. Coefficients of variation ranged from 2.8 to 6.1% for perfusate and from 1.7 to 12.6% for tissue. Recoveries for all compounds exceeded 90%. We applied this method to rat lung tissue, lung perfusate, and rat and human blood. Advantages of this method are simultaneous quantitation with excellent sensitivity of all compounds, simplified peak identification by using multiwavelength detection, and improved accuracy by preventing loss of compounds with metal chelators.