Conversion of leucine to isovaleric (iV) and isocaproic (iC) acids by cell-free extracts of Clostridium bifermentans was demonstrated using two lysis procedures. Sonication resulted in an extract which had the enzymes to convert leucine to alpha-ketoisocaproic acid (alpha-kiC) and thence iV, but failed to produce iC. Extracts prepared by osmotic lysis, which contained intact membranes, could convert leucine to both iV and iC. The enzyme which converts leucine to alpha-kiC was solubilized during osmotic lysis, whereas the decarboxylase and leucine reductase system remained membrane bound. Osmotic lysis also released at least two small molecular weight, heat-stable, anionic components (3500 greater than molecular weight greater than or equal to 1000), which stimulated decarboxylase activity.