A mechanistic mathematical model for carbon oxidation, nitrogen removal, and enhanced biological phosphorus removal was used to develop the Step Bio-P process, a new biological phosphorus and nitrogen removal process with a step-feed configuration. A 9 000-L pilot plant with diurnally varying influent process loading rates was operated to verify the model results and to optimize the Step Bio-P process for application at the Lethbridge, Alberta, Canada, wastewater treatment plant. The pilot plant was operated for 10 months. An automatic on-line data acquisition system with multiple sampling and metering points for dissolved oxygen, mixed liquor suspended solids, ammonia–nitrogen, nitrate–nitrogen, ortho-phosphate, and flow rates was used. A sampling program to obtain off-line data was carried out to verify the information from the on-line system and monitor additional parameters. The on-line and off-line data were used to recalibrate the model, which was used as an experimental design and process optimization tool.The pilot plant results matched and verified the initial mathematical model results. Total phosphorus concentrations were reduced from 14 mg P/L in the biological process influent to less than 1 mg P/L in the secondary effluent. Complete nitrification and 85% nitrogen removal were consistently achieved, and sludge with excellent settling characteristics (sludge volume index values of approximately 80 mL/g) was produced. The step-feed configuration allows an increased bioreactor suspended solids inventory to be maintained while, at the same time, reducing the solids loading rate to the secondary clarifiers. The result is maximum use of existing facilities. The Step Bio-P process is particularly suited for retrofitting conventional activated-sludge plants with limited secondary clarifier capacity to achieve biological nutrient removal (BNR). The combined mathematical modeling and pilot testing program demonstrated that the primary factors affecting the performance of the Step Bio-P process are flow-split percentages, mean cell residence time, dissolved oxygen set points, and short chain volatile fatty acid addition. Process performance is relatively insensitive to anoxic recycle and return activated-sludge flow rates. The growth of excessive quantities of glycogen accumulating organisms occurred when an industrial wastewater with high sugar concentrations was treated. Their growth adversely affected the biological phosphorus removal efficiency of the process. Strategies for controlling growth of these organisms were evaluated and tested.