To provide a comprehensive understanding of the field effect in silicon nanowire (SiNW) sensors, we take a systematic approach to fine tune the distance of a charge layer by controlling the hybridization sites of DNA to the SiNW preimmobilized with peptide nucleic acid (PNA) capture probes. Six target DNAs of the same length, but differentiated successively by three bases in the complementary segment, are hybridized to the PNA. Fluorescent images show that the hybridization occurs exclusively on the SiNW surface between the target DNAs and the PNA. However, the field-effect response of the SiNW sensor decreases as the DNA (charge layer) moves away from the SiNW surface. Theoretical analysis shows that the field effect of the SiNW sensor relies primarily on the location of the charge layer. A maximum of 102% change in resistance is estimated based on the shortest distance of the DNA charge layer (4.7 Å) to the SiNW surface. © 2008 American Chemical Society.