The production of spiramycin by Streptomyces ambofaciens is controlled by nitrogen and carbon sources present in the complex medium containing dextrins, corn oil, and yeast extract as main substrates. Yeast extract appears to be the most important constituent of the culture medium for the production of spiramycin. We show that the assimilated fraction varies in the function of medium composition and increases with the exclusion of dextrins. The yeast extract amino acids (32%) can be used as either a carbon or nitrogen source. It was shown that all amino acids were actively used by the strains during the intensive growth phase. The specific production rate of spiramycin was stimulated by 76% when dextrins were removed from the complex medium containing yeast extract and corn oil as the main compounds. The specific uptake rate of yeast extract amino acids was determined in both cultures (the presence or absence of dextrins). In the presence of dextrins, glutamate, aspartate, asparagine, serine, threonine, alanine, arginine, phenylalanine, histidine, and valine were slowly metabolized whereas isoleucine, leucine, lysine, glycine, and proline were highly assimilated. When dextrins were removed from the medium, the specific uptake rate of the first group of amino acids was increased, whereas the incorporation of the second group was unaffected. The specific uptake rate of tyrosine, methionine, and tryptophane was reduced. A correlation between an enhancement of spiramycin production, the consumption of yeast extract, and amino acids and proteolytic activity was established. This was probably due to the supply of energy source and precursors of spiramycin biosynthesis.