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Activated sludge models can be very useful for designing and managing wastewater treatment plants (WWTPs). However, as with every model, they need to be calibrated for correct and reliable application. Activated sludge model calibration is still a crucial point that needs appropriate guidance. Indeed, although calibration protocols have been developed, the model calibration still represents the main bottleneck to modelling. This paper presents a procedure for the calibration of an activated sludge model based on a comprehensive sensitivity analysis and a novel step-wise Monte Carlo-based calibration of the subset of influential parameters. In the proposed procedure the complex calibration issue is tackled both by making a prior screening of the most influential model parameters and by simplifying the problem of finding the optimal parameter set by splitting the estimation task into steps. The key point of the proposed step-wise procedure is that calibration is undertaken for sub-groups of variables instead of solving a complex multi-objective function. Moreover, even with this step-wise approach parameter identifiability issues may occur, but this is dealt with by using the general likelihood uncertainty estimation (GLUE) method, that so far has rarely been used in the field of wastewater modelling. An example from a real case study illustrates the effectiveness of the proposed methodology. Particularly, a model was built for the simulation of the nutrient removal in a Bardenpho scheme plant. The model was successfully and efficiently calibrated to a large WWTP in Sicily. [ABSTRACT FROM AUTHOR]