Power generation in Microbial fuel cells (MFCs) is a function of various physico-chemical as well as biological parameters. In this study, we have examined the effect of ionic strength, cation exchanger and inoculum age on power generation in a mediator MFC with methylene blue as electron mediator using Enterobacter cloacae IIT-BT08. The effect of ionic strength was studied using NaCl in the anode chamber of a two chambered salt-bridge MFC at concentrations of 5 mM, 10 mM and 15 mM. Maximum power density of 12.8 mW/m(2) was observed when 10 mM NaCl was used. Corresponding current density was noted to be 35.5 mA/m(2). Effect of cation exchanger was observed by replacing salt-bridge with a proton exchange membrane of equal surface area. When the salt-bridge was replaced by a proton exchange membrane, a 3-fold increase in the power density was observed. Power density and current density of 37.8 mW/m(2) and 110.3 mA/m(2) respectively were detected. The influence of the pre-inoculum on the MFC was studied using E. cloacae IIT-BT08 grown for 12, 14, 16 and 18 h. It was observed that 16 h grown culture when inoculated in the anode chamber gave the maximum power output. Power density and current density of 68 mW/m(2) and 168 mA/m(2) respectively were obtained. We demonstrate from these results that both physico-chemical as well as biological parameters need to be optimized for improving the power generation in MFCs. (C) 2009 Published by Elsevier Ltd on behalf of International Association for Hydrogen Energy.