Mitochondria sequester N-methyl-D-aspartate (NMDA)-induced Ca2+ loads and regulate the shape of intracellular Ca2+ concentration ([Ca2+]i) responses in neurons. When isolated mitochondria are exposed to high [Ca2+], Ca2+ enters the matrix via the uniporter and returns to the cytosol by Na+/Ca2+ exchange. Released Ca2+ may re-enter the mitochondrion recycling across the inner membrane dissipating respiratory energy. Ca2+ recycling, the continuous uptake and release of Ca2+ by mitochondria, has not been described in intact neurons. Here we used single-cell microfluorimetry to measure [Ca2+]i and mitochondrially targeted aequorin to measure matrix Ca2+ concentration ([Ca2+]mt) to determine whether Ca2+ recycles across the mitochondrial inner membrane in intact neurons following treatment with NMDA. We used ruthenium red and CGP 37157 to block uptake via the uniporter and release via Na+/Ca2+ exchange, respectively. As predicted by the Ca2+ recycling hypothesis, blocking the uniporter immediately following challenge with 200 μM NMDA produced a rapid and transient increase in cytosolic Ca2+ without a corresponding increase in matrix Ca2+. Blocking mitochondrial Ca2+ release produced the opposite effect, depressing cytosolic Ca2+ levels and prolonging the time for matrix Ca2+ levels to recover. The Ca2+ recycling hypothesis uniquely predicts these reciprocal changes in the Ca2+ levels between the two compartments. Ca2+ recycling was not detected following treatment with 20 μM NMDA. Thus Ca2+ recycling across the inner membrane was more pronounced following treatment with a high relative to a low concentration of NMDA, consistent with a role in Ca2+-dependent neurotoxicity.