The optimal fermentation conditions for the production of pactamycin, a new antitumor antibiotic, by Streptomyces pactum var. pactum were investigated. The optimal pH range for growth was 6.5 to 7.0. The optimal temperature for the growth of the culture and the production of the antibiotic was investigated in a medium containing Cerelose, blackstrap molasses, Pabst yeast, Kay Soy, CaCO(3), and KCl. Since maximal growth and maximal production efficiency was obtained at 32 C, all subsequent fermentations were conducted at this temperature. Pactamycin was bound to the mycelium in different amounts, depending on the fermentation conditions, and could be extracted with acetone. Good yields (216 mug/ml) of pactamycin could be obtained in a medium containing Cerelose, soy-peptone, calcium carbonate, and potassium chloride. Analysis of the biochemical changes during fermentation indicated that pactamycin was produced during the later autolytic phase.