Modulation of voltage-gated calcium channels was studied in inner hair cells (IHCs) in an ex vivo preparation of the apical turn of the rat organ of Corti. Whole-cell voltage clamp in the presence of potassium channel blockers revealed inward calcium currents with millisecond activation and de-activation kinetics. When temperature was raised from 22 to 37 °C the calcium currents of immature IHCs (less than 12 days postnatal) increased 3-fold in amplitude, and developed more pronounced inactivation. This was determined to be calcium-dependent inactivation (CDI) on the basis of its reliance on external calcium (substitution with barium), sensitivity to internal calcium-buffering, and voltage dependence (reflecting the calcium driving force). After the onset of hearing at P12, IHC calcium current amplitude, and the extent of inactivation were greatly reduced. Although smaller than in pre-hearing IHCs, CDI remained significant in the mature IHC near the resting membrane potential. CDI in mature IHCs was enhanced by application of the endoplasmic calcium pump blocker, benzo-hydroquinone. Conversely, CDI in immature IHCs was reduced by calmodulin inhibitors. Thus, voltage-gated calcium channels in mammalian IHCs are subject to a calmodulin-mediated process of CDI. The extent of CDI depends on the balance of calcium buffering mechanisms, and may be regulated by calmodulin-specific processes. CDI provides a means for the rate of spontaneous transmitter release to be adjusted to variations in hair cell resting potential and steady state calcium influx.