Hypoxia impairs brain function by incompletely defined mechanisms. Mild hypoxia, which impairs memory and judgment, decreases acetylcholine (ACh) synthesis, but not the levels of ATP or the adenylate energy charge. However, the effects of mild hypoxia on the synthesis of the glucose-derived amino acids [alanine, aspartate, gamma-amino butyric acid (GABA), glutamate, glutamine, and serine] have not been characterized. Thus, we examined the incorporation of [U-14C]glucose into these amino acids and ACh during anemic hypoxia (injection of NaNO2), hypoxic hypoxia (15 or 10% O2), and hypoxic hypoxia plus hypercarbia (15 or 10% O2 with 5% CO2). In general, the synthesis of the amino acids and of ACh declined in parallel with each type of hypoxia we studied. For example, anemic hypoxia (75 mg/kg of NaNO2) decreased the incorporation of [U-14C]glucose into the amino acids and into ACh similarly. [Percent inhibition: ACh (57.4), alanine (34.4), aspartate (49.2), GABA (61.9), glutamine (59.2), glutamate (51.0), and serine (36.7)]. A comparison of several levels (37.5, 75, 150, 225 mg/kg of NaNO2) of anemic hypoxia showed a parallel decreased in the flux of glucose into ACh and into the amino acids whose synthesis depends on mitochondrial oxidation: GABA (r = 0.98), glutamate (r = 0.99), aspartate (r = 0.96), and glutamine (r = 0.97). The synthesis of the amino acids not dependent on mitochondrial oxidation did not correlate as well with changes in ACh metabolism: serine (r = 0.68) and alanine (r = 0.76). The decreases in glucose incorporation into ACh and into the amino acids with hypoxic hypoxia (15% or 10% O2) or hypoxic hypoxia with 5% CO2 were very similar to those with the two lowest levels of anemic hypoxic. Thus, and explanation of the brain's sensitivity to a decrease in oxygen availability must include the alterations in the metabolism of the amino acid neurotransmitters as well as ACh.