Isolated guinea-pig outer hair cells (OHCs) (n=52) were inserted into a partitioning microchamber and electromotility was measured by a calibrated optoelectronic apparatus. Acetylcholine (ACh), and ACh together with different protein kinase inhibitors, were applied to OHCs through a puffer pipette. ACh produced a magnitude increase of electromotility. This magnitude increase was inhibited by co-application of KN-62, a calcium/calmodulin-dependent protein kinase II (CAMKII) inhibitor. Simultaneous application of ACh and H-89, a selective protein kinase A (PKA) inhibitor, did not antagonize the ACh response. Further support for the CAMKII-mediated ACh influence on electromotility is that the magnitude increase is also inhibited by the calmodulin antagonist trifluoperazine (TFP) and by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) inhibitor thapsigargin. The results suggest an essential role of calcium in the ACh-mediated increase of the magnitude of electromotility. Elevation of the intracellular calcium concentration apparently activates CAMKII which, in turn, phosphorylates membrane or cytoskeletal substrate(s). This molecular modification probably leads to reduced axial cell stiffness and subsequent increase of the electromotile response.