We have developed a novel microrespirometric method to characterize the inhibitory effects due to heavy metals on activated sludge treatment. This method was based on pulse dynamic respirometry and involved the injection of several pulses of substrate and inhibitors, of increasing concentration. Furthermore, we evaluated the inhibitory effects of heavy metals (copper and zinc), substrate and biomass concentrations, and pH on activated sludge activity. While higher biomass concentrations counteracted the inhibitory effects of both copper and zinc, higher substrate concentrations predominantly augmented the inhibitory effect of copper but no significant change in inhibition by zinc was observed. pH had a clear but relatively small effect on inhibition, partially explained by thermodynamic speciation. We determined the key kinetic parameters; namely, the half saturation constant (K S ) and the maximum oxygen uptake rate (OUR max ). The results showed that higher heavy metal concentrations substantially decreased K S and OUR max suggesting that the inhibition was uncompetitive.