The method of studying intracellular free Ca2+ ([Ca2+]i) in outer hair cells (OHC) of guinea pig using digital imaging microscopy and Ca(2+)-sensitive fluorescent dye Fura-2 was introduced. [Ca2+]i can be calculated from fluorescence intensity ratios with excitation lights of two different wave-lengths, using Fura-2 loaded in OHC. The data showed that acetylcholine (ACh) can induce [Ca2+]i elevation of OHC in the solution with calcium. Mechanism of ACh inducing [Ca2+]i elevation and application of calcium imaging microfluorometry for studying cochlea hair cell were discussed.