Vestibular hair cells were isolated from the macula utriculi, macula sacculi and crista ampullaris of the guinea pig, using enyzmatic and mechanical techniques. The cells could be classified into two types: flask-shaped ones with a neck and rod- or round-shaped ones without a neck. The flask-shaped cells were thought to be type I cells, the rod-shaped cells type II cells, while the round-shaped cells could have been type I or type II cells. The intracellular free calcium ion concentrations ([Ca2+]i) of these cells were determined using the Ca2+-sensitive dye Fura-2 and digital imaging miroscopy. In the resting state, [Ca2+]i in the nucleus and cuticular plate was variable in both types of cells. The 150 mM KCl stimulation, which might induce a depolarization, resulted in a temporary increase in [Ca2+]i in both types of cells. In the presence of 1 µm ionomycin, there was an irreversible increase in [Ca2+]i in both types of cells. These observations aid in elucidating vestibular function.