The aminoglycoside antibiotic gentamicin elicits proximal tubular toxicity and cell death. In calcium-sensing receptor (CaR)-transfected HEK-293 (CaR-HEK) cells and CaR-expressing proximal tubule-derived opossum kidney (OK) cells, chronic gentamicin treatment elicits dose-dependent, caspase-mediated apoptotic cell death. Here we investigated whether the renal cell toxicity of the CaR agonist gentamicin could be prevented by CaR antagonism or by lithium cotreatment which may interfere with receptor-mediated signalling. Chronic treatment of OK and CaR-HEK cells with low concentrations of gentamicin elicited cell death, an effect that was ameliorated by cotreatment with the CaR negative allosteric modulator (calcilytic) NPS-89636. This calcilytic also attenuated CaR agonist-induced ERK activation in these cells. In addition, 1 mM LiCl, equivalent to its therapeutic plasma concentration, also inhibited gentamicin-induced toxicity in both cell types. This protective effect of lithium was not due to the disruption of phosphatidylinositol-mediated gentamicin uptake as the cellular entry of Texas red-conjugated gentamicin into OK and CaR-HEK cells was unchanged by lithium treatment. However, the protective effect of lithium was mimicked by glycogen synthase 3beta inhibition. Together, these data implicate CaR activation and a lithium-inhibitable signalling pathway in the induction of cell death by gentamicin in renal epithelial cells in culture.