Low oxygen (hypoxia) decreases the release and synthesis of acetylcholine (ACh) as well as behavioral performance (i.e., tight rope test scores). These hypoxic-induced impairments are partially reversed by 3,4-diaminopyridine (3,4-DAP). With mouse brain slices, the Ca++-dependent-K+-stimulated synthesis of ACh from [U-14C]glucose decreased 45% under 2.5% oxygen and 80% under 0% oxygen. During hypoxia, 3,4-DAP (10 nM) increased in vitro ACh synthesis 67% (2.5% oxygen) and 63% (0% oxygen). The Ca++-dependent-K+-stimulated release of ACh declined by 55 or 67% under 2.5 or 0% oxygen, respectively.3,4-DAP partially reversed this hypoxic impaired release of ACh under 2.5 (+69%) or 0% (+226%) oxygen. In vivo, chemical hypoxia (i.e., NaNO2-induced methemoglobinemia) reduced ACh synthesis from [U-14C]glucose in striatum (-82%), cortex (-68%) and hippocampus (-55%). During hypoxia, 3,4-DAP (10 pmol/kg) increased [U-14C]glucose incorporation into ACh in the hippocampus (+62%) and striatum (+36%). Tight rope test performance decreased 89% during hypoxia and 3,4-DAP diminished this decline to only 55%. These findings demonstrate that the hypoxic-induced deficits in ACh metabolism and behavior can be partially reversed by interaction with Ca++ homeostasis. The therapeutic usefulness of 3,4-DAP in the treatment of human metabolic encephalopathies remains to be evaluated.