Ca2+ signaling serves distinct purposes in different parts of a hair cell. The Ca2+ concentration in stereocilia regulates adaptation and, through rapid transduction-channel reclosure, underlies amplification of mechanical signals. In presynaptic active zones, Ca2+ mediates the exocytotic release of afferent neurotransmitter. At efferent synapses, Ca2+ activates the K+ channels that dominate the inhibitory postsynaptic potential. A copious supply of diffusible protein buffer isolates the three signals by restricting the spread of free Ca2+ and limiting the duration of its action. Using cDNA subtraction and a gene expression assay based on in situ hybridization, we detected abundant expression of mRNAs encoding the Ca2+ buffer parvalbumin 3 in bullfrog saccular and chicken cochlear hair cells. We cloned cDNAs encoding this protein from the corresponding inner-ear libraries and raised antisera against recombinant bullfrog parvalbumin 3. Immunohistochemical labeling indicated that parvalbumin 3 is a prominent Ca2+-binding protein in the compact, cylindrical hair cells of the bullfrog's sacculus, and occurs as well in the narrow, peanut-shaped hair cells of that organ. Using quantitative Western blot analysis, we ascertained that the concentration of parvalbumin 3 in saccular hair cells is approximately 3 mM. Parvalbumin 3 is therefore a significant mobile Ca2+ buffer, and perhaps the dominant buffer, in many types of hair cell. Moreover, parvalbumin 3 provides an early marker for developing hair cells in the frog, chicken, and zebrafish.