We have optimized a method for measuring total hydroxyproline (HYP) in urine by HPLC after release from urinary peptides by solid-phase hydrolysis on Dowex 50W x 8 ion-exchange resin. The HYP was derivatized with 4-chlor-7-nitrobenzo-2-oxa-1,3-diazole, and excess reagent was removed with the use of a 100-mg C18 Bond-Elut cartridge. The HYP derivative was separated isocratically at 30 degrees C on a 250 x 4.6 mm reversed-phase column containing 5-microns particles of Spherisorb S5 ODS-2, with S-carboxymethylcysteine as internal standard. Total assay time was 14 min. The standard curve for the method was linear from the detection limit for HYP, 3.6 mumol/L, to 10 mmol/L. The between-batch CV was less than 5.1% and the mean analytical recovery of HYP was 95% +/- 1.4%. Comparison with a commercially available colorimetric method showed good correlation: y = 1.158x + 19.76 mumol/L (Syx = 74, n = 120), but HPLC results were 15% higher, probably from incomplete hydrolysis with the colorimetric method. This method offers a considerable improvement in assay time, specificity, sensitivity, precision, and cost compared with the colorimetric method.