Phenylalanine hydroxylase (PAH) is a pterin-dependent non-heme metalloenzyme that catalyzes the oxidation of phenylalanine to tyrosine, which is the rate-limiting step in the catabolism of Phe. Chromobacteriumviolaceum phenylalanine hydroxylase (cPAH) has been prepared and its steady-state mechanism has been investigated. The enzyme requires iron for maximal activity. Initial rate measurements, done in the presence of the 6,7-dimethyl-5,6,7,8-tetrahydropterin (DMPH4) cofactor, yielded an average apparent kcat of 36±1 s–1. The apparent KM values measured for the substrates DMPH4, L-Phe, and O2 are 44±7, 59±10, and 76±7 µM, respectively. Steady-state kinetic analyses using double-reciprocal plots revealed line patterns consistent with a sequential ter-bi mechanism in which L-Phe is the middle substrate in the order of binding. The occurrence of a line intersection on the double-reciprocal plot abscissa when either pterin or O2 is saturated suggests that, prior to O2 binding, DMPH4 and L-Phe are in associative pre-equilibrium with cPAH. Together with an inhibition study using the oxidized cofactor, 7,8-dimethyl-6,7-dihydropterin, it is conclusive that the mechanism is fully ordered, with DMPH4 binding the active site first, L-Phe second, and O2 last. This represents the first conclusive steady-state mechanism for a PAH enzyme. Electronic supplementary material is available if you access this article at http://dx.doi.org/10.1007/s00775-002-0395-6. On that page (frame on the left side), a link takes you directly to the supplementary material.