Cochlear hair cells are presumed to live in culture for many days, yet they are difficult to identify in cultured tissues. We stained hair cells in cochlear sections with FM1-43 and cultured them in collagen matrix. Three rows of outer hair cells and a single row of inner ones were distinguished by staining with FM1-43. Fixation of the sections with paraformaldehyde caused loss of the FM1-43 fluorescence, indicating that FM1-43 stained only live hair cells. In sections cultured for 48 h, almost all hair cells were still positive with FM1-43. Culture with gentamycin caused loss of FM1-43-positive cells. h. serum-free, long-term cultures (15 days) performed without antibiotics or neurotrophins, the row alignment of FM1-43-positive hair cells was still maintained. Membranous labyrinth-like vacuoles enveloping hair cells were formed in the collagen matrix. Accordingly, FM1-43 is an efficient marker for identifying live hair cells in cultured tissues. Moreover, cochlear hair cells are revealed to live for weeks in serum-free culture without exogenous neurotrophins. (C) 2003 Elsevier Ireland Ltd. All rights reserved.