We describe a method for determining urinary riboflavin by "high-pressure" liquid chromatography, with fluorometric detection. An aliquot of a 24-h urine specimen is injected directly into the chromatograph and the natural fluorescence of riboflavin is measured as the compound is eluted. Interference from other fluorophores is obviated because of differing retention times. Urinary components other than riboflavin and its analogs or degradation products exhibit no significant fluorescence at the wavelengths used (450 nm excitation, 530 nm emission). Analytical recovery of added riboflavin was 96.5 (SD 1.1)%. The CV within-run was 0.7%, between-run it was 4.3%. Concentrations of riboflavin as low as 10 micrograms/L are readily detected, with a linear relation of response to concentration to at least 2000 micrograms/L.