INTRODUCTION: Cochlea supporting cells are primary targets for inducing hair cell regeneration as they are part of the stem/progenitor cell (SPC) population and are also the primary expression site of connexin 26 (Cx26), a gap junction protein, which plays a role in the maintenance of the endocochlear potential. Mutations in the GJB2 gene, which encodes Cx26, are the most common cause of nonsyndromic-inherited deafness in humans. Therefore, studies on Cx26 may help elucidate not only the inner ear physiology, but also the regeneration potential of its supporting cells. OBJECTIVE: Investigate the expression of connexin 26 in suspension cultures of a dissociated organ of Corti SPC from mice. METHODS: Cultures were maintained for 2 days in vitro (2DIV) in a medium. The primary antibodies, connexin 26 (1:100) (Zymed), musashi (1:100) (Abcam), vimentin (1:100) (Abcam), and secondary antibodies, Alexa Fluor 488 and 546 (1:400) (Invitrogen), were used for indirect immunofluorescence of the culture cells (2DIV) with DAPI for nuclei staining. Musashi and vimentin are markers of SPC and cell cycle division, respectively. RESULTS: In the present study, we demonstrated that in vitro otospheres express Cx26 within 2 days, besides other established markers, musashi and vimentin. Different studies have shown that distinct human connexin paralogs are expressed in human embryonic stem (ES) cells. CONCLUSION: Although further phenotypic characterization of otosphere cells is necessary, we demonstrated for the first time that Cx26 is expressed in SPC cultured from the organ of Corti of mice that supports the hypothesis that the connexin protein family may be functional in pluripotency maintenance.