The H/D exchange reaction between isobutane and acidic USY zeolite was studied using mass spectrometry and in situ H-1 NMR. An inhibiting effect of hydrogen on this reaction was observed. The HID exchange is enhanced by both in situ and ex situ addition of small amounts of isobutene. A kinetic isotope effect of 1.5 was found for the H/D exchange of isobutane and 2-deuterium-labeled isobutane over D2O-exchanged HUSY (DUSY), as well as nondeuterated and perdeuterated isobutane over HUSY. The occurrence of a primary kinetic isotope effect (KIE) indicates that the slow step of the H/D exchange reaction involves a cleavage of the tertiary C-H (or C-D) bond in isobutane. After addition of small amounts of isobutene to the feed or pretreating the catalyst with small amounts of isobutene before starting the H/D exchange reaction, the KIE still exists, suggesting that the limiting step for the H/D exchange reaction between isobutane and acidic zeolite is the hydride transfer from an alkane to the exchanged cation.