Stereocilin is defective in a recessive form of deafness, DFNB16. We studied the distribution of stereocilin in the developing and mature mouse inner ear and analyzed the consequences of its absence in stereocilin-null (Strc−/−) mice that suffer hearing loss starting at post-natal day 15 (P15) and progressing until P60. Using immunofluorescence and immunogold electron microscopy, stereocilin was detected in association with two cell surface specializations specific to outer hair cells (OHCs) in the mature cochlea: the horizontal top connectors that join the apical regions of adjacent stereocilia within the hair bundle, and the attachment links that attach the tallest stereocilia to the overlying tectorial membrane. Stereocilin was also detected around the kinocilium of vestibular hair cells and immature OHCs. In Strc−/− mice, the OHC hair bundle was structurally and functionally normal until P9. Top connectors, however, did not form and the cohesiveness of the OHC hair bundle progressively deteriorated from P10. The stereocilia were still interconnected by tip links at P14, but these progressively disappeared from P15. By P60, the stereocilia, still arranged in a V-shaped bundle, were fully disconnected from each other. Stereocilia imprints on the lower surface of the tectorial membrane were also not observed in Strc−/− mice, thus indicating that the tips of the tallest stereocilia failed to be embedded into this gel. We conclude that stereocilin is essential to the formation of horizontal top connectors. We propose that these links, which maintain the cohesiveness of the mature OHC hair bundle, are required for tip-link turn over.