To evaluate uridine levels in humans we developed a very sensitive and specific high-performance liquid chromatographic method for the determination of uridine in serum. We use techniques which are available in a standard analytical laboratory. Chromatographic analysis was carried out on a Phenomenex Aqua C18 5 micro 125A column protected by a guard cartridge system. Potassium dihydrogen phosphate buffer-acetonitrile was used as an eluent and oxypurinol as the internal standard. All sample preparation steps were done at 4 degrees C and the autosampler was cooled down to 4 degrees C. The calibration curve was linear throughout the calibration range from 0.25 to 100 micromol/l. This method was primarily established to evaluate uridine serum levels in patients with HIV infection since patients on highly active antiretroviral therapy (HAART) might develop metabolic disturbances that could lead to severe and fatal lactic acidosis due to mitochondrial toxicity. It is suggested that a limited or inadequate uridine supply is at least in part responsible for the onset of such deterioration.