The presence of glycogen in the central nervous has been known for many years, but assigning a physiological role has been difficult. Recent experiments in adult rodent optic nerve have shown that glycogen supports axonal conduction during periods of decreased glucose availability and increased energy demand under normoglycaemic conditions (Brown et al., 2003). It is accepted that glycogen is metabolized to lactate in astrocytes, and released into the extracellular space, for uptake by axons. Although lactate has been measured in the hundreds of micromolar range in the brain extracellular space (Hu & Wilson, 1997), the dynamics of lactate release and the relative contribution of glycolysis and glycogenolysis are unknown. In this study we used lactate biosensors placed against the optic nerve to measure real-time lactate levels in response to a variety of metabolic interventions. We used enzyme-based sensors to measure lactate release from adult mouse optic nerve, while simultaneously recording the stimulus evoked compound action potential (CAP), to correlate axon conduction with lactate concentration ([lactate]o). Recordings were carried out in vitro in a superfusion chamber at 37°C. The basal [lactate]o recorded from nerves perfused with 10 mM glucose aCSF was 410.2 ± 28.3 μM (n = 18), implying continuous release of lactate under resting conditions. The [lactate]o did not change when glucose was increased above 5 mM, but decreased with [glucose] of 2, 1 and 0.5 mM. Glucose withdrawal led to a rapid fall in [lactate]o but a delayed fall in the CAP (Brown et al., 2003), confirming glucose as the source of lactate. Both the [lactate]o and CAP were rapidly abolished in the presence of the glycolytic inhibitor iodoacetate, implying an absolute requirement for glycolytic metabolism of glucose for both axon conduction and lactate production. In the presence of the glycogen phosphorylase inhibitor DAB [lactate]o decreased by 22.2 ± 1.6 % (n = 3) indicating glycogen metabolism contributes to the lactate signal. Imposing a 10 kHz stimulus on the nerve resulted in a 14.7 ± 2.3 % increase in [lactate]o with subsequent DAB introduction resulting in a fall of 33.9 ± 2.0 % (n = 3) in [lactate]o, implying that increased tissue energy demand activates glycogenolysis, such that the contribution of glycogen-derived lactate to the [lactate]o increases relative to tissue energy demand. These data indicate that mouse optic nerve continually releases lactate into the extracellular space. The source of lactate is via both metabolism of glucose and glycogen, but the contribution of glycogen-derived lactate increases in line with tissue energy demand.