The determination of lactate dynamics in brain tissue represents a challenge, partly because in vivo data at cellular resolution are not available. Here we monitored lactate in astrocytes and neurons of the primary somatosensory cortex of mice using the genetically-encoded FRET sensor Laconic in combination with two-photon laser scanning microscopy. An intravenous lactate injection rapidly increased the Laconic signal in both astrocytes and neurons, demonstrating high lactate permeability across the tissue. The signal increase was significantly smaller in astrocytes pointing to higher basal lactate levels in these cells, confirmed by a one-point in vivo calibration protocol.Trans-acceleration of the monocarboxylate transporter with pyruvate was able to reduce intracellular lactate in astrocytes but not in neurons. Collectively, this data provides in vivo evidence for a lactate gradient from astrocytes to neurons.