Active processes in the inner ear of lizards can be monitored using spontaneous otoacoustic emissions (SOAE) measured outside the eardrum. In the Australian bobtail lizard, SOAE are generated by an active motility process in the hair-cell bundle. This mechanism has been shown to be sensitive to the calcium-chelating agent BAPTA and is presumed to be related to the calcium-sensitive transduction-channel motor implicated in other non-mammalian hair cell systems. In studies of frog saccular and turtle auditory papillar hair cells in vitro, the frequency and amplitude of bundle oscillations depend on the concentration of calcium in the bathing solutions. In the present study, the calcium concentration in the endolymph was changed in vivo in the Australian bobtail lizard Tiliqua rugosa, and SOAE were monitored. Glass pipettes with large tips and containing different calcium concentrations in their fluids were introduced into scala media and their contents allowed to passively flow into the endolymph. Low calcium concentrations resulted in a downward shift in the frequency of SOAE spectral peaks and generally an increase in their amplitudes. Calcium concentrations above 2mM resulted in increases in frequency of SOAE peaks and generally a loss in amplitude. These frequency shifts were consistent with in vitro data on the frequencies and amplitudes of spontaneous oscillation of hair cell bundles, and thus also implicate calcium ions in the generation of active motility in non-mammalian hair cells. The data also suggest that in this lizard species, the ionic calcium concentration in the cochlear endolymph is at least 1mM.