Xanthurenic acid (XA) has been quantified in the serum of normal and vitamin B6-deficient rabbits using high performance liquid chromatography. The concentration of XA in the serum of normal and B6-deficient rabbits was 141 and 2275 ng/ml, respectively. The coefficient of variation for a series of dilutions of standard XA (3.9 to 1000 ng) ranged from 45.5% at the lower limit of the curve to 10.9% at the higher range of the curve. The minimum detectable level was 3.9 ng/ml. Serum samples spiked with reference XA exhibited a parallel dose response. The percentage recovery of XA from serum samples was 80.8%. The procedure, which requires 1 to 2 ml of serum, is sensitive and may be a useful tool for assessing B6 nutritional parameters as well as the physiological role of XA. It offers advantages over urinary procedures because it is more sensitive, more specific, and allows the study of blood levels of XA.