Clinical gentamicin use is associated with proximal tubular toxicity and we have shown that chronic gentamicin treatment elicits dose-dependent, caspase-mediated cell death in proximal tubule-derived opossum kidney (OK) cells and calcium-sensing receptor (CaR)-transfected HEK-293 (CaR-HEK) cells (1). The CaR localises to the apical membrane of the proximal tubule and is sensitive to gentamicin (2,3). Therefore, we investigated whether renal gentamicin toxicity could be attenuated either by CaR antagonism or by the use of the inositol phosphate signalling modulator lithium. OK and CaR-HEK cells were grown for 4-7 days in 1% foetal bovine serum-containing media and gentamicin cellular toxicity was investigated by Trypan Blue uptake or by flow cytometry (FACS). Chronic treatment of OK and CaR-HEK cells with concentrations of gentamicin (20-50μM) similar to those employed clinically, induced cell death after 6-7 days, consistent with the time course of clinical nephrotoxicity. Cotreatment with the calcilytic NPS-89636 (1μM; CaR negative allosteric modulator) abolished CaR agonist-induced ERK activation in OK and CaR-HEK cells. NPS-89636 also attenuated 100μM gentamicin-induced cell death (-77%, P<0.05 by ANOVA) in OK cells, whilst abolishing 200μM gentamicin-induced cell death in CaR-HEK cells, thus implicating the CaR in these responses. There is evidence that LiCl protects rats from gentamicin nephrotoxicity. To determine whether the LiCl exerts its effect directly on renal cells, we studied gentamicin-induced cell death in both OK cells and CaR-HEK cells in the presence or absence of a therapeutic concentration of LiCl (1mM; 4 days) and found that it also inhibited gentamicin toxicity in both cell types. The decrease in gentamicin toxicity was not due to decreased cellular uptake as equivalent levels of texas red-conjugated gentamicin were found in OK and CaR-HEK cells in the presence or absence of LiCl. Similarly, lithium failed to significantly alter acute gentamicin-induced Ca²⁺_i mobilisation. However, the cyto-protective effect of lithium was mimicked by the glycogen synthase kinase-3 inhibitors IX and XI. Together, these data implicate the CaR in the mediation of gentamicin toxicity in OK and CaR-HEK cells. In addition, under culture conditions, lithium cotreatment ameliorates renal cell aminoglycoside toxicity but without preventing the cellular uptake of the drug.