We developed an HPLC method for the measurement of inorganic phosphate using thymidine phosphorylase (EC 220.127.116.11). This enzyme catalyzes the phosphorolysis of thymidine to 2-deoxyribose 1-phosphate and thymine. Thymine release was measured at 265 nm after separation by reverse-phase HPLC. The assay was sensitive enough to detect as little as 10 pmol of Pi. The response to the phosphate concentration was linear from 1 to 100 microM. The value of this method was demonstrated in an analysis of the kinetics of Pi release from PPi in the presence of Catharanthus roseus tonoplast pyrophosphatase.