The solute carrier transmembrane protein prestin (SLC26A5) drives an active electromechanical transduction process in cochlear outer hair cells that increases hearing sensitivity and frequency discrimination in mammals. A large intramembraneous charge movement, the nonlinear capacitance (NLC), is the electrical signature of prestin function. The transmembrane domain (TMD) helices and residues involved in the intramembrane charge displacement remain unknown. We have performed cysteine-scanning mutagenesis with serine or valine replacement to investigate the importance of cysteine residues to prestin structure and function. The distribution of oligomeric states and membrane abundance of prestin was also probed to investigate whether cysteine residues participate in prestin oligomerization and/or NLC. Our results reveal that 1) Cys-196 (TMD 4) and Cys-415 (TMD 10) do not tolerate serine replacement, and thus maintaining hydrophobicity at these locations is important for the mechanism of charge movement; 2) Cys-260 (TMD 6) and Cys-381 (TMD 9) tolerate serine replacement and are probably water-exposed; and 3) if disulfide bonds are present, they do not serve a functional role as measured via NLC. These novel findings are consistent with a recent structural model, which proposes that prestin contains an occluded aqueous pore, and we posit that the orientations of transmembrane domain helices 4 and 10 are essential for proper prestin function.