BACKGROUND Low concentration of plasma pyridoxal-5-phosphate (PLP) is associated with hyperhomocysteinemia and inflammation. Most methods for the measurement of plasma PLP require large specimen volume and involve the use of toxic reagents. METHODS We have developed a HPLC method for the measurement of PLP and 4-pyridoxic acid (4-PA) in plasma, which requires small specimen volume. The samples are prepared without adding any toxic reagents. Furthermore, we have examined whether intake of vitamin B6 affects the concentration of plasma PLP and 4-PA. RESULTS The coefficient of variation of the method was 6% and the recovery of the added vitamin in plasma was about 100%. The concentrations of plasma PLP and 4-PA in 168 healthy subjects were 40.6 (8.4-165.0) nmol/L, median and (range) and 17.5 (3.7-114.79) nmol/L, median and (range) respectively. In the multiple regression analyses, the concentration of plasma PLP was associated with the concentration of plasma 4-PA (p<0.0001), BMI, (p=0.02) and sex, (p=0.0008). The concentration of plasma 4-PA was associated with plasma PLP (p<0.0001), serum folate (p=0.004), smoking (p=0.03) and vitamin B6 intake (p=0.01). CONCLUSIONS The present method is suitable for large clinical studies for the measurement of plasma PLP and 4-PA. Our findings demonstrate that plasma 4-PA, BMI and sex are the major determinants of plasma PLP in healthy individuals.