Flat carbon anodes placed near a cathode in a microbial fuel cell (MFC) are adversely affected by oxygen crossover, but graphite fiber brush anodes placed near the cathode produce high power densities. The impact of the brush size and electrode spacing was examined by varying the distance of the brush end from the cathode and solution conductivity in multiple MFCs. The startup time was increased from 8 ± 1 days with full brushes (all buffer concentrations) to 13 days (50 mM), 14 days (25 mM) and 21 days (8 mM) when 75% of the brush anode was removed. When MFCs were all first acclimated with a full brush, up to 65% of the brush material could be removed without appreciably altering maximum power. Electrochemical impedance spectroscopy (EIS) showed that the main source of internal resistance (IR) was diffusion resistance, which together with solution resistance reached 100 Ω. The IR using EIS compared well with that obtained using the polarization data slope method, indicating no major components of IR were missed. These results show that using full brush anodes avoids adverse effects of oxygen crossover during startup, although brushes are much larger than needed to sustain high power. © 2011 Elsevier B.V.