The calcium ionophore ionomycin has been shown to induce length increases of guinea pig outer hair cells (). We have demonstrated that these length increases can be inhibited by a 30 min preincubation of the cells with the protein kinase inhibitor ML-9. At either 30 or 60 s after ionomycin application, the effect of ML-9 was dose-dependent with a half maximal response at approximately 0.3 μM. No effect on cell length was detected after 30 min incubation with 0.5 and 5 μM ML-9 alone. However, with 50 and 500 μM ML-9, significant contraction in cell length was observed. 50 μM ML-9 did not interfere with the ability of ionomycin to elevate fluorescence of the calcium indicator Fluo-3, nor did it alter the ability of cells to exclude propidium iodide from their nuclei. Treatment with 500 μM ML-9 resulted in impaired cell morphology. The data support the hypothesis that protein kinase activity regulates calcium-dependent processes that effect shape changes of outer hair cells. They are consistent with the involvement of the calcium/calmodulin-dependent enzyme, myosin light chain kinase, a known target of ML-9, but do not preclude the possibility of another intracellular target for ML-9.