The removal of benzene, toluene, ethylbenzene and xylene (BTEX) as quaternary mixtures were studied in batch systems using a well-defined mixed microbial culture. The synergistic and antagonistic effects of total BTEX removal (BTEXT-RE) due to the presence of mixed substrate was evaluated through experiments designed by response surface methodology (RSM). The low and high concentrations of individual BTEX were 15 and 75 mg l(-1), respectively. The results showed that, increasing the concentration of xylene increased the cumulative BTEX removal (BTEXT-RE), however the reverse occurred when benzene concentrations were increased from low to high levels. A mixed response of increasing and decreasing trend in the BTEXT-RE value was observed when either of toluene or ethylbenzene concentration was increased. When the concentrations of individual BTEX compounds were 30 mg l(-1), the BTEXT-RE was about 58%. Complete BTEXT-RE was achieved at optimal BTEX concentrations of 48.1, 45.6, 49.3 and 56.6 mg l(-1). The RSM approach was found efficient in explaining the main, squared and interaction effects among individual BTEX concentrations on the BTEXT-RE in a more statistically meaningful way.