SUMMARY: The formation of acetoin was studied with 44 strains of the genus Acetobacter, when growing on a medium containing DL-lactate as the main carbon source. Most of the strains produced only limited amounts of acetoin. However, strains of the species A. rancens, pasteurianum and ascendens converted most of their substrate into acetoin, up to 74% of the theoretical amount. The influence of the substrate concentration and of the degree of aeration was studied. The Voges-Proskauer positive substance was isolated from large-scale fermentations, purified and definitely identified as acetoin. It was present chiefly in the laevo-rotatory form. Young resting cells oxidized d(---)- lactate, L(+)- lactate, sodium pyruvate, acetoin and diacetyl, often nearly to completion. Cell-free extracts synthesized acetoin from pyruvate optimally at about pH 7. More than 99% of the enzyme activity was located in the soluble enzyme fraction. Thiamin pyrophosphate stimulated CO2 and acetoin production from pyruvate. Evidence is presented that acetoin may arise both from the acetaldehyde and from the α-acetolactate pathway.