New York City Environmental Protection in conjunction with City College of New York assessed the application of the anammox process in the reject water treatment using a moving bed biofilm reactor (MBBR) located at the 26th Ward wastewater treatment plant, in Brooklyn, NY. The single-stage nitritation/anammox MBBR was seeded with activated sludge and consequently was enriched with its own ‘homegrown’ anammox bacteria (AMX). Objectives of this study included collection of additional process kinetic and operating data and assessment of the effect of nitrogen loading rates on process performance. The initial target total inorganic nitrogen removal of 70% was limited by the low alkalinity concentration available in the influent reject water. Higher removals were achieved after supplementing the alkalinity by adding sodium hydroxide. Throughout startup and process optimization, quantitative real-time polymerase chain reaction (qPCR) analyses were used for monitoring the relevant species enriched in the biofilm and in the suspension. Maximum nitrogen removal rate was achieved by stimulating the growth of a thick biofilm on the carriers, and controlling the concentration of dissolved oxygen in the bulk flow and the nitrogen loading rates per surface area; all three appear to have contributed in suppressing nitrite-oxidizing bacteria activity while enriching AMX density within the biofilm.