In 1941 Krampitz and Werkman (1) discovered an enzyme in Micro- coccus lysodeikticus which catalyzed the decarboxylation of oxalacetate. By means of an exchange reaction they were able to demonstrate the in- corporation of isotopic carbon dioxide into the p-carboxyl group of oxal- acetic acid in the presence of this enzyme (2). Similar results were ob- tained by Evans, Vennesland, and Slotin (3) with an enzyme system from pigeon liver. Further studies on the C+Cl condensation have been re- viewed by Wood (4) and will, therefore, not be discussed here. The apparent importance of oxalacetate decarboxylase as one of the gateway enzymes for the fixation of carbon dioxide into compounds of the Krebs isocitric acid cycle made it of interest to attempt to purify this enzyme and to study some of its properties. The biotin content of the enzyme fractions at various stages of purity was determined in view of the recent implication of biotin in the metabolism of carbon dioxide (5-11). Lee, Burris, and Wilson (12) demonstrated that cell-free preparations of Azotobacter vinelandii have oxalacetate decarboxylase activity. Since this organism is easily grown on a large scale (13), cell-free extracts of this bacterium were used as the source material for this study.