Kinetic isotope effects (KIES) were measured for methyl glucoside (4) hydrolysis on unlabeled material by NMR. Twenty-eight C-13 KlEs were measured on the acid-catalyzed hydrolysis of alpha-4 and beta-4, as well as enzymatic hydrolyses with yeast (x-glucosidase and almond P-glucosidase. The 1-C-13 KIES on the acid-catalyzed reactions of alpha-4 and beta-4, 1.007(2) and 1.010(6), respectively, were in excellent agreement with the previously reported values (1.007(1), 1.011(2): Bennet and Sinnott, J. Am. Chem. Soc. 1986, 108, 7287). Transition state analysis of the acid-catalyzed reactions using the C-13 KIES, along with the previously reported 2 H KIES, confirmed that both reactions proceed with a stepwise D-N*A(N) mechanism and showed that the glucosyl oxocarbenium ion intermediate exists in an E-3 sofa or H-4(3) half-chair conformation. C-13 KIES showed that the alpha-glucosidase reaction also proceeded through a DN*AN mechanism, with a 1-C-13 KIE of 1.010(4). The secondary C-13 KIES showed evidence of distortions in the glucosyl ring at the transition state. For the beta-glucosidase-catalyzed reaction, the 1-C-13 KIE of 1.032(1) demonstrated a concerted A(N)D(N) mechanism. The pattern of secondary C-13 KIES was similar to the acid-catalyzed reaction, showing no signs of distortion. KIE measurement at natural abundance makes it possible to determine KIES much more quickly than previously, both by increasing the speed of KIE measurement and by obviating the need for synthesis of isotopically labeled compounds.