Abstract This paper presents a methodology for the optimization of the operation of an alternating oxidation ditch system, performing carbon oxidation–nitrification, denitrification and settling, through the use of a mathematical model and pilot testing. Measurements have been made in a pilot plant located in the Sanitary Engineering Research and Development Center of E.Y.D.A.P at Metamorphosis-Athens. Also, a mathematical model consisting of 1-dimensional mass balance (convection–dispersion) equations, based on the IAWPRC activated sludge model No. 1 was used. After the calibration and verification of the model with the experimental data, the optimization of the system’s performance was achieved by testing operational cycles with different time lengths. Since carbon removal is way under the 91/271 E.U. directive’s upper limits and since the ditch system has not been designed for phosphorous removal, the limits for nitrogen removal have been used for comparison. In this work’s example, from the eight oper...