PURPOSE: In the receptor organs of the inner ear, hair cells detect mechanical stimuli such as sounds and accelerations by deflection of their hair bundles. Myosin regulatory light chain (RLC) and non-muscle myosin II (NM2) are expressed at the apical surfaces of hair cells, and NM2 and the phosphorylation of RLC by myosin light chain kinase (MLCK) have earlier been shown to regulate the shapes of hair cells' apical surfaces in rodents. The aim of our study was to elucidate the function of myosin molecules on hair cell physiology. METHODS: We investigated the expression of NM2 and RLC in the bullfrog's saccule by immunostaining. Using NM2 and MLCK inhibitors, we measured the stiffness, spontaneous oscillation, and resting open probability of frog hair bundles. Six to ten saccules from pleural animals were used in each experiment. In addition, we recorded auditory brainstem responses in ten mice after transtympanic injection of an MLCK inhibitor. RESULTS: We confirmed the expression of NM2A/B and MYL9 on the apical surfaces of hair cells and of NM2A and MYL12A in hair bundles. We found that NM2 and MLCK inhibitors reduce the stiffness of hair bundles from the bullfrog's saccule. Moreover, MLCK inhibition inhibits the spontaneous oscillation of hair bundles and increases the resting open probability of transduction channels. In addition, MLCK inhibition elevates hearing thresholds in mice. CONCLUSION: We conclude that NM2 and the phosphorylation of RLC modulate the physiological function of hair cells and thereby help to set the normal operating conditions of hair bundles.