Steady-state kinetic properties of purified 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. strain P.J. 874 were examined by a 14CO2 method at pH 7.5 and 37 degrees C. The results conform to a mono-iso-ordered bi-bi mechanism with binding of 4-hydroxyphenylpyruvate before O2 and release of CO2 before homogentisate. A Theorell-Chance mechanism can not be excluded. The apparent DV and DV/Km ratios were about 1.1 and 1.2, respectively, for 4-hydroxy[2,6-2H2]phenylpyruvate when the initial O2 consumption was measured. The stoichiometry between the consumption of O2 and the formation of homogentisate and CO2 was 1:1:1. Loss of hydrogen at C2 or C6 of the ring of the substrate is thus not the rate-limiting step during the reaction. Instead, this appears to be the conversion of an isomerized enzyme form.