Enhanced biological phosphorus removal (EBPR) requires an anaerobic-aerobic sequence and short chain fatty acids, namely acetate. It is also known that the presence of nitrate in the anaerobic phase inhibits EBPR. This study describes a lab-scale experimentation carried out to study the effect of different substrates on EBPR and behaviour of PAOs under anoxic conditions in a sequencing batch reactor operated using synthetic wastewater. Experimental data show that the EBPR performance is significantly affected by glucose rich influent. Low COD/TKN ratios caused lower phosphorus removal performance since nitrate entering the anaerobic zone consumes substrate for denitrification. The results also show that anoxic phosphate uptake took place together with nitrate reduction when there was no external substrate. However, the uptake rate under anoxic conditions was lower than that under aerobic conditions.