Confocal laser scanning microscopy permits detailed visualization of structures deep within thick fluorescently labeled specimen. This makes it possible to investigate living cells inside intact tissue without prior chemical sample fixation and sectioning. Isolated guinea pig temporal bones have previously been used for confocal experiments in vitro, but tissue deterioration limits their use to a few hours after the death of the animal. In order to preserve the cochlea in an optimal functional and physiological condition, we have developed an in vivo model based on a confocal microscopy approach. Using a ventral surgical approach, the inner ear is exposed in deeply anaesthetized, tracheotomized, living guinea pigs. To label the inner ear structures, scala tympani is perfused via an opening in the basal turn, delivering tissue culture medium with fluorescent vital dyes (RH 795 and calcein AM). An apical opening is made in the bony shell of cochlea to enable visualization using a custom-built objective lens. Intravital confocal microscopy, with preserved blood and nerve supply, may offer an important tool for studying auditory physiology and the pathology of hearing loss. After acoustic overstimulation, shortening and swelling of the sensory hair cells were observed.