Free ammonia has long been identified as a nitrite oxidation inhibitor. However, past attempts to use this compound to eliminate nitrite oxidation and thereby promote more efficient nitrogen removal strategies during biological wastewater treatment have often failed. Additionally, contradictory results exist in the literature where direct measurements of free ammonia inhibition of nitrite oxidation have been reported. In this study, suspended biomass samples (nitrifier enriched activated sludge) were collected from a bench scale nitrification reactor with Nitrobacter spp. as the dominant nitrite oxidizer and subjected to batch respirometric experiments designed to quantify free ammonia inhibition of nitrite oxidization. A variety of data including ammonia, nitrite, and nitrate conversion rates, oxygen consumption rates, and Nitrobacter ribosomal RNA transcript abundance, a molecular indicator of growth activity, were used to assess nitrite oxidation and growth activity. Both the traditional and molecular activity assessments indicated that free ammonia had a limited inhibitory effect on Nitrobacter spp. In fact, the pH changes necessary to induce high free ammonia concentrations (>10 mg-NIL) had a demonstrably more important inhibiting effect on nitrite oxidation than free ammonia. In contrast, during high ammonia oxidizing activity (5.3 mg-NIL/h), low nitrite oxidation rates (0.2 mg-N/L/h) and severely impaired Nitrobacter spp. growth activity, indicated by a low abundance of the Nitrobacter spp. ribosomal gene transcript relative to the ribosomal gene (0.08), were measured. The findings suggest that pH changes and ammonia oxidizing bacteria activity are more important factors limiting Nitrobacter spp. mediated nitrite oxidation, rather than the free ammonia concentration. (C) 2010 Elsevier Ltd. All rights reserved.