({ }^{13}) C NMR approach was taken to study the biotransformation of ((R)) - and ((S)-\left[2-{ }^{13} \mathrm{C}, 2-{ }^{2} \mathrm{H}\right]) ibuprofen-CoA in isolated rat liver mitochondria. The ({ }^{13} \mathrm{C}) chemical shift induced by ({ }^{2} \mathrm{H}) substitution for the (\mathrm{C}-2) of (\left[2{ }^{13} \mathrm{C}, 2-{ }^{2} \mathrm{H}\right]-) ibuprofen-CoA is about (0.5 \mathrm{ppm}) relative to the corresponding ' (\mathrm{H})-bearing thioester. The (\mathrm{C}-2) resonances for (\left[2-{ }^{13} \mathrm{C}\right]-) and (\left[2-{ }^{13} \mathrm{C}, 2-{ }^{2} \mathrm{H}\right]) ibuprofen-CoA are (5 \mathrm{ppm}) downfield with respect to their acid counterparts. Consequently, this technique permits real time analyses of the metabolic fate of the doubly labeled substrate in a quantitative manner. The initial rate of the epimerization of ((S)-\left[2-{ }^{13} \mathrm{C}, 2-{ }^{2} \mathrm{H}\right]) ibuprofen-CoA relative to the ((R))-counterpart by intact mitochondria was estimated to be 1.5 , which is in line with the equilibrium constant determined with the purified epimerase. Contrary to the reports by other groups, this ({ }^{13} \mathrm{C}) NMR study did not find spontaneous hydrolysis of the CoA-thioester upon being exposed to intact mitochondria. Current focus of this investigation is to extend this NMR methodology to the levels of whole cells and live animals to gain a better understanding of the enantioselective metabolism of ibuprofen. ΒΏΔ 1993 Academic Press, Inc.