The astrocyte-neuronal lactate-shuttle hypothesis posits that lactate released from astrocytes into the extracellular space is metabolized by neurons. The lactate released should alter extracellular pH (pHe), and changes in pH in central chemosensory regions of the brainstem stimulate ventilation. Therefore, we assessed the impact of disrupting the lactate shuttle by administering 100 M ␣-cyano-4-hydroxycinnamate (4-CIN), a dose that blocks the neuronal monocarboxylate transporter (MCT) 2 but not the astrocytic MCTs (MCT1 and MCT4). Administration of 4-CIN focally in the retrotrapezoid nucleus (RTN), a medullary central chemosensory nucleus, increased ventilation and decreased pHe in intact animals. In medullary brain slices, 4-CIN reduced astrocytic intracellular pH (pHi) slightly but alkalinized neuronal pHi. Nonetheless, pHi fell significantly in both cell types when they were treated with exogenous lactate, although 100 M 4-CIN significantly reduced the magnitude of the acidosis in neurons but not astrocytes. Finally, 4-CIN treatment increased the uptake of a fluorescent 2-deoxy-D-glucose analog in neurons but did not alter the uptake rate of this 2-deoxy-D-glucose analog in astrocytes. These data confirm the existence of an astrocyte to neuron lactate shuttle in intact animals in the RTN, and lactate derived from astrocytes forms part of the central chemosensory stimulus for ventilation in this nucleus. When the lactate shuttle was disrupted by treatment with 4-CIN, neurons increased the uptake of glucose. Therefore, neurons seem to metabolize a combination of glucose and lactate (and other substances such as pyruvate) depending, in part, on the availability of each of these particular substrates.