Oral thymine loading tests were performed on 12 healthy volunteers. Each subject was given a single oral dose of 250 mg thymine in capsule form. Blood, urine and saliva samples were collected pre-dose and up to 5 h post-dose. Concentrations of thymine, and its catabolites dihydrothymine and ß-ureidoisobutyrate were analysed by HPLC-tandem mass spectrometry in plasma, urine and saliva. The pharmacokinetic data of healthy volunteers were analysed assuming a non-compartmental model. Thymine peaked quickly (30–45 min) in plasma to a maximum concentration of 170 ± 185 μg/L (mean ± SD). Clearance was high (mean 57.9 L/h/kg) exceeding normal human liver blood flow, suggesting low systemic bioavailability; urinary recovery of the thymine dose was low (< 1%). Apparent formation rate-limited kinetics were observed for dihydrothymine, and the plasma concentration of dihydrothymine was consistently 10-fold higher than that of thymine. Plasma ß-ureidoisobutyrate concentrations, on the other hand, were similar to that of thymine. Genotyping confirmed that pathological mutations of the DPYD gene were absent. The urinary excretion ratio of thymine/dihydrothymine was informative of the maximum concentration. Saliva thymine was highly variable. These data are potentially useful as a basis for developing of a screening procedure to prospectively identify patients who are at risk of toxicity from fluoropyrimidine drugs.