The molecular basis of the close linkage between oxidative metabolism and acetylcholine (ACh) synthesis is still unclear. We studied this problem in slices and synaptosomes by measurement of ACh synthesis from [U-14C]glucose, and 14CO2 production from [3,4-14C]- and [2-14C]glucose, an index of glucose decarboxylation by the pyruvate dehydrogenase complex (PDH) and the enzymes of the Krebs cycle, respectively. We examined both under conditions that either inhibited (low O2 or antimycin) or stimulated (2,4-dinitrophenol [DNP] or 35 mM-K+) 14CO2 production from [2-14C]- or [3,4-14C]glucose. Incorporation of [U-14C]glucose into ACh was reduced under low O2 and by antimycin or DNP (by 51--93%) and stimulated by 35 mM-K+ (by 30--60%). Under all of these conditions, ACh synthesis and the decarboxylation of [3,4-14C]- and [2-14C]glucose were linearly related (r = 0.741 and 0.579, respectively). The difference in the rate of 14CO2 production from [3,4-14C]- and [2-14C]glucose was used as a measure of the amount of glucose that was not oxidatively decarboxylated (efflux). We found that efflux was reduced (low O2 and antimycin), unchanged (DNP in slices), or increased (DNP in synaptosomes and K+ stimulation in slices) compared with control values under 100% O2. ACh synthesis and efflux were more closely related (r = 0.860) than ACh synthesis and 14CO2 production from variously labeled glucoses.