Substrate inhibition of 2,4-denitrophenol (DNP) degradation was investigated using activated sludge which had been adapted to mineralize DNP. DNP is a metabolic uncoupler, preventing cells from making energy for growth and it has been suggested that pH may be important in mitigation effects of uncouplers. After acclimation of the activated sludge, the effect of pH on toxicity of DNP at high concentration (75 mg/L) was investigated, over a pH range of 5 to 9. DNP inhibition was found to be strongly dependent on mixed liquor pH. The DMP degradation rate was highest in the pH range of 6.95 to 7.84; at pH 5.94 degradation of 75 mg/L DNP was significantly inhibited; at pH < 5.77, DNP degradation was completely inhibited after approximately 30% of the DNP was degraded. By comparison, no significant effect of pH variation in the same range was seen on glucose uptake by the activated sludge culture.