An HPLC method for measuring total hydroxyproline in human urine was validated. Hydroxyproline derivatization was achieved with 9-fluorenylmethyl chloroformate after blocking primary amino acids with orthophthaldialdehyde. The derivatives (hydroxyproline and internal standard) were separated by reversed phase high-performance liquid chromatography and detected by absorbance at 254 nm. Duplicate measurements of hydroxyproline have a coefficient of variation of 3.9% and the recovery in spiked urine samples is between 99.5 and 100.8%. We have compared the HPLC procedure with a commercial colorimetric assay. Analytical criteria of these methods are identical. Regression analysis, involving 50 samples, shows an excellent correlation between hydroxyproline chromatographic (y) and colorimetric (x) procedures: y = 0.989x + 2.99 (r = 0.976). Hydroxyproline excretion was determined in urine samples from 76 women more than 5 years post-menopause. The mean hydroxyproline/creatinine ratio in this group was 19.3 +/- 5.6 mumol/mmol (range 10.6-34.7). Finally, we compared in the same urinary samples hydroxyproline excretion with pyridinoline excretion (hydroxylysylpyridinoline and lysylpyridinoline), a new marker of bone resorption. The values show a significant correlation, with r = 0.417 for hydroxylysylpyridinoline and r = 0.443 for lysylpyridinoline.