Nitrobacter is an autotrophic soil bacterium that derives its entire energy supply from the reaction NO2-+ O2 = NO3-+17 kcal. Apart from the early studies of Meyerhof (1916 a, b), there has been little biochemical work done on this organism since its discovery and isolation by Winogradsky (1890, 1891). The present paper records investigations into the mechanism of niitrite oxidation by Nitrobacter. Preliminary notes on some of our results have already appeared (Lees & Simpson, 1955a, b). MATERIALS AND METHODS Growth of the organisms. Nitrobacter was obtained from an Aberdeen garden soil by serial subculture in a medium made up as follows. To 10 1. of water were added 30 g. of KH2P04, 1 g. of CaSO4, 0 5 g. of MgCl2, 0 01 g. of MnCl2, 0-2 ml. of dialysed iron (B.P.). By addition of lOM-NaOH, the pH was adjusted to 7-8, which is the optimum pH for the strain of Nitrobacter used (Lees, 1954). The solution was filtered and, after addition of the appropriate amount of NaN02, sterilized at 1200 for 20 min. This medium (medium G) was used also for the propagation of the stock cultures which provided inocula for large-scale batch cultures. For largescale batch cultures 100 ml. of the carbonate-phosphate suspension, used by Lees (1952) for the culture of Nitrosomonas, was added to each 101. of medium G before sterilization. Batch cultures were used for preparing suspensions of Nitrobacter cells suitable for biochemical study; the carbonate-phosphate suspension was added to facilitate the separation of these cells after growth. Nitrobacter, unulike Nitrosomonas, does not require a solid surface for growth, but Nitrobacter cells did adhere in some measure to the solid particles in the suspension and, in any case, were entrained by them when the cells were separated from the bulk of the medium by centrifuging.