Expansion of a CAG repeat in the Huntington's disease (HD) gene results in progressive neuronal loss, particularly of striatal medium-sized spiny neurons (MSNs). Studies in human HD autopsy brain tissue, as well as cellular and animal models of HD, suggest that increased activity of NMDA-type glutamate receptors and altered mitochondrial function contribute to selective neuronal degeneration. In this regard, the YAC128 mouse model, expressing full-length human huntingtin with 128 glutamine repeats, has been the focus of much interest. Although NMDA-induced apoptosis is enhanced in YAC128 MSNs, here we report that the initial steps in the death signaling pathway, including NMDA receptor (NMDAR) current and cytosolic Ca2+ loading, are similar to those observed in wild-type MSNs. In contrast, we found that the NMDAR-mediated Ca2+ load triggered a strikingly enhanced loss of mitochondrial membrane potential in YAC128 MSNs, suggesting that NMDAR signaling via the mitochondrial apoptotic pathway is altered. This effect was accompanied by impaired cytosolic Ca2+ clearance after removal of NMDA, a difference that was not apparent after high potassium-evoked depolarization-mediated Ca2+ entry. Inhibition of the mitochondrial permeability transition (mPT) reduced peak cytosolic Ca2+ and mitochondrial depolarization evoked by NMDA in YAC128 MSNs but not wild-type MSNs. Hence, in contrast to YAC models with moderate CAG expansions, the enhanced NMDA-induced apoptosis in YAC128 MSNs is predominantly determined by augmented mitochondrial sensitivity to Ca2+-induced activation of the mPT. These results suggest that the CAG repeat length influences the mechanism by which mHtt enhances NMDAR-mediated excitotoxicity.