Background: The amino acid transporter xCT (SLC7A11) is a sodium independent, chloride dependent exchanger which must form a heterodimer with 4F2hc (SLC3A2) in order to function. xCT mediates the exchange of glutamate and cystine. Cystine is essential for the synthesis of the antioxidant glutathione and it has been suggested that xCT activity is a rate limiting factor for glutathione synthesis [1]. N-acetylcysteine (NAC) is reported to be a substrate for xCT and due to its higher solubility has been used in place of cystine for physiological studies. However it is not clear how it was demonstrated that NAC is a substrate for xCT. Therefore the aim of this investigation was to investigate whether or not NAC is a substrate for human xCT expressed in Xenopus oocytes. Methods: Xenopus laevis oocytes were detached and defolliculated in oocyte Ringer’s 2 buffer, transferred to ND91 buffer and incubated at 18° C overnight before being microinjected with xCT-GFP and 4F2hc cRNA and incubated for a further 48 hours. To determine relative 14c-glutamate uptake rates injected and non-injected oocytes were incubated with 20 µM 14c-glutamate for 5 min before uptake was stopped with 1 ml ice cold ND91 and washed twice. Uptake of 14c-glutamate into oocytes was determined by liquid scintillation counting. To demonstrate efflux by exchange batches of ten injected oocytes (3 batches/condition/experiment) were incubated with 20 µM 14c-glutamate for 30 min at room temperature to preload the oocytes. Oocytes were then washed twice in ND91 buffer and incubated with ND91 buffer alone or 10 mM glutamate, 10 mM NAC, a saturated solution of cystine or 10 mM glycine for five min. The media was removed and efflux of 14c-glutamate determined in a liquid scintillation counter. Parallel experiments were carried out in non-injected oocytes. Data are median fold change from stated control groups (range). Results: 14c-glutamate uptake into injected oocytes was 1.9 (±0.29) fold higher compared to non-injected controls (n=3). Compared to oocytes incubated with ND91 alone, the efflux of 14c-glutamate into the media was 5.1 (3.3-8.8) fold higher for NAC (n=3 ovaries), 3.4 (1.8-4.4) fold higher for glutamate (n=2) and 4.1 fold higher for cystine (n=1) while glycine (n=2) did not appear to stimulate efflux with a fold change of 1.0 (1.01-1.02). In non-injected oocytes NAC, glutamate, cystine or glycine did not stimulate efflux of 14c-glutamate. Conclusions: xCT/4F2hc injected oocytes showed increased uptake of 14c-glutamate and efflux of 14c-glutamate was observed in response to NAC, glutamate and cystine but not to glycine which is not an xCT substrate. This study demonstrates that NAC is a substrate for xCT.