A high-performance liquid chromatographic (HPLC) method has been developed for measuring thymidine and thymine levels, so these could be used to determine thymidine phosphorylase (TP) activity. The method described has the selectivity to separate the analytes from an enzymatic reaction mixture without mutual disturbance. Correlation of peak areas of thymine, thymidine, and 2-thio-6-azauridine with amounts in standards and spiked matrices was linear in the concentration range 0.04–2 μg mL−1 for 2-thio-6-azauridine, 0.08–3 μg mL−1 for thymine, and 0.02–2.5 μg mL−1 for thymidine. Intra- and interday precision were measured by analyzing the retention times of replicate standards or spiked matrices within one (intra assay,n=8) and eight consecutive days (inter assay,n=8) over the concentration ranges 0.1–0.8 μg mL−1 for thymine, 0.02–0.4 μg mL−1 for thymidine, and 0.1–0.5 μg mL−1 for 2-thio-6-azauridine. Recovery was monitored by use of 2-thio-6-azauridine as internal standard. The precision, accuracy, and speed of the method were excellent. The smallest detectable (LOD) and quantifiable (LOQ) quantities were, respectively, 1.3 ng mL−1 and 3.4 ng mL−1 for thymine, 2.68 ng mL−1 and 7.46 ng mL−1 for thymidine, and 1.7 ng mL−1 and 4.92 ng mL−1 for 2-thio-6-azauridine. At 25°C the TP reaction was linear for at least 45 min. At 37°C the reaction occurred much faster and was linear for 15 min only. We decided, therefore, to quantify thymidine and thymine after an incubation time of 30 min at 25°C. The Michaelis constant (K M=59 nmol) and maximum enzymatic reaction velocity (v max=1.626 nmol min−1×106 cells) for TP were determined by extrapolation of the regression line (y=32.53x+90.73;r=0.992) to thex andy axes, respectively. Enzymatic activity was calculated from: