A complex organic medium had been developed for this organism, but a chemically defined medium was not available. A number of carbohydrates were tested and it was determined that pentoses, glucose, starch, and dextrine produced the best antibiotic yields. A combination of glucose and dextrine was chosen for routine use. Use of five amino acids: L-asparagine, glycine, L-alanine, L-leucine and L-histidine gave the highest antibiotic yields. High levels of leucine were tolerated by the organism and were in some instances very helpful to antibiotic yields, but no single amino acid was essential and various combinations could be used. It was not necessary to add vitamins to the medium and fatty acids were not beneficial, but magnesium, calcium, iron, potassium, and phosphate were required for growth and antibiotic production. A chemically defined medium was evolved which supported good growth of the organism and the production of 5000-6000 mcg/ml of capreomycin.