Intracellular isoforms of the enzyme Ca-ATPase were identified in the inner ear by immunostaining paraffin sections with a polyclonal antiserum against rabbit cardiac muscle Ca-ATPase. In the adult cochlea, intense staining was present at the lateral border of outer hair cells in regions corresponding with the distribution of the subsurface cisternal system. Other cell types containing high levels of Ca-ATPase were skeletal muscle fibers in the tensor tympani, vascular smooth muscle, spiral ganglion neurons and subpopulations of fibrocytes in the limbus, spiral ligament and underlying vestibular neurosensory epithelium. In neonatal gerbils, staining of tensor tympani muscle fibers was observed at 4 days after birth and approached adult levels by 8 days after birth. Ca-ATPase was first detected in other cell types between postnatal days 12 and 14 but immunostaining still remained well below the intensity seen in adults at 20 days after birth. The demonstration of abundant calcium pumps in the subsurface cisternae confirms the role of this organelle as an intracellular reservoir for Ca2+ in outer hair cells. The presence of high levels of Ca-ATPase in spiral ganglion neurons and in fibrocytes specialized for ion transport points to a role for the enzyme in regulating the activity of other cell types of importance to normal hearing.