Cisplatin, a commonly used antineoplastic agent, destroys the sensory hair cells in the cochlear and vestibular system leading to irreversible hearing loss and balance problems. Cisplatin-induced hair cell damage presumably occurs by apoptosis. Recent studies suggest that p53 may play an important role initiating cisplatin-induced apoptosis in some cell types. To determine if p53 plays a role in cisplatin-mediated hair cell loss, cochlear and utricular organotypic cultures were prepared from postnatal day 3-4 rats and treated with cisplatin or cisplatin plus pifithrin-alpha (PFT), a p53 inhibitor. Control cultures were devoid of p53 immunolabeling, caspase-1 and caspase-3 labeling and p53 protein was absent from Western blots. Cisplatin (1-10 microg/ml) caused a dose-dependent loss of hair cells in cochlear and utricular cultures, up-regulated phospho-p53 serine 15 immunolabeling, increased the expression of phospho-p53 serine 15 in Western blots from 6 to 48 h after the onset of cisplatin-treatment, and increased caspase-1 and caspase-3 labeling in cochlear and vestibular cultures. Addition of PFT (20-100 microM) to cisplatin-treated cochlear and utricular cultures resulted in a dose-dependent increase in hair cell survival; suppressed the expression of p53 in Western blots and eliminated caspase-1 and caspase-3 labeling in cultures. These results suggest that the tumor suppressor protein, p53, plays a critical role in initiating apoptosis in cochlear and vestibular hair cells. Temporary suppression of p53 with PFT provides significant protection against cisplatin-induced hair cell loss and offers the potential for reducing the ototoxic, vestibulotoxic and neurotoxic side effects of cisplatin.