Ifosfamide (IF) nephrotoxicity is a serious adverse effect in children undergoing chemotherapy. Previous studies have shown that, in addition to the renal production of chloroacetaldehyde, a toxic metabolite of IF, lower levels of glutathione (GSH) may predispose the kidney to damage. The antioxidant N-acetylcysteine (NAC) is used extensively as an antidote for acetaminophen poisoning in children by replenishing GSH levels. As it has been safely and effectively used clinically, the objective of this study was to test whether the reversal of ifosfamide-induced nephrotoxicity can be achieved by administering NAC. Supplementation with NAC may reduce or prevent the degree of cellular cytotoxicity induced by IF. Porcine renal proximal tubular (LLCPK-1) cells were treated with NAC (0.4 mM or 2.5 mM) concurrently with 1 mM IF and 50 microM L-buthionine sulfoximine (BSO). Cellular viability was assessed by alamarBlue assay at 96 h. Intracellular GSH and oxidized GSH (GSSG) levels were determined using a GSH/GSSG colorimetric detection kit. A significant 60% decrease in cellular viability occurred when cells were treated daily with BSO and IF for 96 h. This decrease was significantly reduced when cells were concurrently treated with NAC in a concentration-dependent manner. Intracellular and total GSH levels in cells receiving concurrent treatment of NAC were significantly higher than those without NAC treatment. NAC protects renal tubular cells from IF-induced cytotoxicity. It is likely that NAC is protecting the cells by partially acting as a precursor for GSH synthesis. This mode of therapy may allow for protecting children from life-threatening nephrotoxicity induced by IF.