Under the Ping-Pong bi-bi mechanism, it is possible to independently study the kinetics of reductive half-reaction (RHR), i.e., binding of the steroid substrate to the enzyme active site, oxidation and reduction of the enzyme's FAD followed by the release of the product, and oxidative half-reaction (OHR), i.e., binding of DCPIP to the active site, oxidation of FADH -to FAD and release of the reduced DCPIPH2. Therefore, we employed pre-steady-state kinetics to investigate the pH dependence of both RHR and OHR. The aim was to explain our previous observation that the enzyme exhibits two reaction pH optima; one at pH 6.5 when the reaction was conducted with DCPIP and the second at pH 8.5 when the reaction was conducted with PMS or PMS and DCPIP . Initially, we hypothesized that under acidic conditions, the reaction catalysed by AcmB might proceed according to a different mechanism from that of the other KstDs. However, later on, we suspected that it is the sluggish OHR that is responsible for the observed pH optimum of the steady-state reaction.