There are few studies in which direct measurement of vitamin B6 status in both plasma and red cells has been assessed. The aims of the present study were to evaluate the use of a simple, robust HPLC method of direct pyridoxal 5'-phosphate (PLP) measurement in plasma and red cells and to assess its use in establishing reference ranges in a healthy population. A reverse phase HPLC method with pre-column derivatisation using semicarbazide for the simultaneous measurement of PLP, its degradation product, 4-pyridoxic acid (PA) and pyridoxal (PL) in plasma and red cells was developed. Pre-column derivatisation, reverse phase chromatography and detection procedures were optimised. The recovery, precision, linearity and sensitivity of the assay for plasma and red cell PLP, PA and PL was established. The recovery of PLP was greater than 95% for both plasma and red cell samples. The Intra and Inter batch imprecision for PLP was less than 6% and 7%, respectively. The method for PLP was linear up to at least 1000 nmol/l and the detection limit was 2.1 nmol/l (limit of quantification; 5.8 nmol/l). Accuracy of PLP measurements in plasma were acceptable, showing a mean bias of 4.5% from the mean value of laboratories (N=34) participating in an external quality assurance scheme. Geometric mean (95% reference intervals) for plasma and red cell PLP in the healthy subjects (N=126) were 56 (21-138) nmol/l and 410 (250-680) pmol/g Hb, respectively. There was a strong positive correlation (r(2)=0.81) between plasma and red cell PLP levels in the reference population. The HPLC method described was found to be suitable for the routine measurement of PLP in both plasma and red cells.