The outer hair cell (OHC) lateral wall is a unique trilaminate structure consisting of the plasma membrane, the cortical lattice, and subsurface cisternae. OHCs are capable of altering their length in response to transmembrane voltage change. This so-called electromotile response is presumed to result from conformational changes of membrane-bound protein molecules, named prestin. OHC motility is accompanied by axial stiffness changes when the membrane potential of the cell is altered. During length changes, intracellular anions (mainly Cl^-) act as extrinsic voltage sensors. In this study, we inquired whether the motor proteins are responsible for the voltage-dependent axial stiffness of OHCs, and whether ACh, the neurotransmitter of efferent neurons, modulates the stiffness of the cortical lattice and/or the stiffness of the motor protein. The experiments were done on isolated guinea pig OHCs in the whole-cell voltage-clamp mode. Axial stiffness was determined by loading a fiber of known stiffness onto the apical surface of the cells. Voltage-dependent stiffness and cell motility disappeared, and the axial stiffness of the cells significantly decreased after removal of intracellular Cl^-. The result suggests that the stiffness of the motor protein is a major contributor to the global axial stiffness of OHCs. ACh was found to affect both the motor protein and other lateral wall stiffness components.