Further insight into the metabolic status of cells within anode biofilms is essential for understanding the functioning of microbial fuel cells and developing strategies to optimize their power output. Cells throughout anode biofilms of Geobacter sulfurreducens reduced the metabolic stains: 5-cyano-2,3-ditolyl tetrazolium chloride and Redox Green, suggesting metabolic activity throughout the biofilm. To compare the metabolic status of cells growing close to the anode versus cells in the outer portion of the anode biofilm, anode biofilms were encased in resin and sectioned into inner (0-20 microm from anode surface) and outer (30-60 microm) fractions. Transcriptional analysis revealed that, at a twofold threshold, 146 genes had significant (P<0.05) differences in transcript abundance between the inner and outer biofilm sections. Only 1 gene, GSU0093, a hypothetical ATP-binding cassette transporter, had significantly higher transcript abundances in the outer biofilm. Genes with lower transcript abundance in the outer biofilm included genes for ribosomal proteins and NADH dehydrogenase, suggesting lower metabolic rates. However, differences in transcript abundance were relatively low (<threefold) and the expression of genes for the tricarboxylic acid cycle enzymes was not significantly lower. Lower expression of genes involved in stress responses in the outer biofilm may reflect the development of low pH near the surface of the anode. The results of this study suggest that cells throughout the biofilm are metabolically active and can potentially contribute to current production. The microtoming/microarray strategy described here may be useful for evaluating gene expression with depth in a diversity of microbial biofilms.