Calcium ion plays an important role in the hair cell's mechanoelectrical transduction process; in particular, Ca2+ controls adaptation to protracted mechanical stimuli. Because calmodulin is a ubiquitous intracellular receptor for Ca2+ and has been shown to accumulate at the tips of stereocilia, we determined its concentration and identified the proteins with which it interacts in the hair bundle. By performing quantitative immunoblot analysis on isolated bundles, we ascertained that the average concentration of calmodulin within each stereocilium is approximately 70 microM. Extraction experiments disclosed that, in the presence of 20 microM Ca2+, 50% of the calmodulin is bound to detergent-soluble receptors. To distinguish these receptors, we developed an assay that utilizes calmodulin crosslinked to alkaline phosphatase. This technique is approximately 100-fold more sensitive than calmodulin-binding assays that employ 125I- or biotin-labeled calmodulin. When used with chemiluminescence detection in a blot-overlay assay, the calmodulin-alkaline phosphatase conjugate identified hair-bundle proteins of molecular masses 25, 35, 145, 175, 240, and 350 kDa. We examined the subcellular distribution of these receptors; all but the 240-kDa molecule are soluble in a nonionic detergent. The relatively high concentration of calmodulin and the presence of several calmodulin-binding proteins provide evidence for a role of calmodulin in hair bundles.