4-Hydroxyphenylpyruvate (HPP) is converted to homogentisate by 4-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) in tyrosine catabolism. The reaction involves oxidative decarboxylation, hydroxylation of the phenyl ring, and substituent migration. We have studied HPPD using rapid mixing pre-steady state kinetic methods in conjunction with steady state kinetic analysis to find evidence for the accumulation and identity of intermediates under pseudo-first order conditions. The global analysis of spectrophotometric data indicates that there are three intermediates that accumulate during a single turnover. From fluorescence experiments in conjunction with rapid mixing acid quench and product analysis it can be established that the product is formed on the enzyme before the completion of turnover and that the primary rate limiting step is product release. We have measured the steady-state and pre-steady state isotope effects with both 2,3,5,6-tetradeuterio-HPP substrate and in the presence of deuterated solvent. Our results suggest that re-aromatization to form the product and its release are the last two processes of turnover. Hereditary type 1 tyrosinemia is a result of a deficiency of the enzyme, fumarylacetoacetase, that catalyzes the final step in tyrosine catabolism. In the absence of treatment, patients who suffer from this disease generally die from either kidney failure or from primary liver cancer. NTBC inhibits HPPD, thereby shutting down four latter steps of tyrosine catabolism and completely alleviating the symptoms of T1Y. Despite numerous studies on NTBC and similar inhibitors, the explicit binding mode was, until recently, unknown. We have crystallized and solved the structure of HPPD in complex with NTBC.