Separate quantification of glutamate (Glu) and glutamine (Gln) using conventional MRS on clinical scanners is challenging. In previous work, constant-time point-resolved spectroscopy (CT-PRESS) was optimized at 3 T to detect Glu, but did not resolve Gln. To quantify Glu and Gln, a time-domain basis set was constructed taking into account metabolite T 2 relaxation times and dephasing from B 0 inhomogeneity. Metabolite concentrations were estimated by fitting the basis one-dimensional CT-PRESS diagonal magnitude spectra to the measured spectrum. This method was first validated using seven custom-built phantoms containing variable metabolite concentrations, and then applied to in vivo data acquired in rats exposed to vaporized ethanol and controls. Separate metabolite quantification revealed increased Gln after 16 weeks and increased Glu after 24 weeks of vaporized ethanol exposure in ethanol-treated compared with control rats. Without separate quantification, the signal from the combined resonances of Glu and Gln (Glx) showed an increase at both 16 and 24 weeks in ethanol-exposed rats, precluding the determination of the independent and differential contribution of each metabolite at each time. Copyright © 2012 John Wiley & Sons, Ltd.