An “endoneurial” preparation from a rabbit tibial nerve fascicle was used to study the ability of peripheral nerve axons and Schwann cells to derive their composite energy requirements from glucose, D-β-hydroxybutyrate, or albumin-bound palmitate, and the effects of insulin in vitro on their composite glucose utilization. Samples incubated with 5 mM glucose for 2 h maintained a stable O₂ uptake and P-creatine and ATP concentrations, and they exhibited a slight increase in P-creatine/creatine ratio (the electron microscopic appearance of the preparation was previously shown to be unaltered under these conditions). The rate of glucose oxidation required to account for the O₂ uptake accounted for 61% of the glucose uptake. In samples incubated without substrate for 2 h, a marked fall in tissue glucose was associated with a 50% decrease in O₂ uptake and with decreases in P-creatine, ATP, and in the P-creatine/creatine ratio. In medium lacking glucose but containing 5 mM DL-β-hydroxybutyrate, a stable rate of D-β-hydroxybutyrate uptake was observed, and acetoacetate production accounted for only a small fraction; significant decreases in O₂ uptake or ATP were prevented, and, although P-creatine and the P-creatine/creatine ratio fell, they remained significantly higher than after incubation without substrate. An efficient blood-nerve barrier to albumin is known to exist. Medium containing albumin-bound palmitate with molar ratios of palmitate/albumin of 1 or 2 (highest FFA concentration, 1.32 meq/L) failed to prevent decreases in P-creatine, ATP, and in the P-creatine/creatine ratio during incubations without glucose; the associated O₂ uptakes suggested that the tissue is susceptible to respiratory uncoupling and depression on exposure to albumin-bound palmitate as compared with non-neural tissue. Insulin (100 or 1000 μU/ml) had no detectable effects on glucose utilization in the endoneurial preparation during 2-h incubations with 5 mM glucose or (U-¹⁴C) glucose. In contrast, in epineurial tissue from rabbit sciatic nerve, insulin (100 μU/ml) increased (U-¹⁴C) glucose incorporation into CO₂ and total lipid. The neural components of peripheral nerve are probably dependent on glucose as their major substrate for energy production and respiration under most physiologic conditions in which elevated plasma ketone body concentrations are absent; their composite glucose utilization is not subject to acute, direct regulation by insulin in concentrations that might reasonably be derived from plasma insulin of pancreatic origin.