A chemically defined medium was developed for the biosynthesis of cephalosporin C by Paecilomycespersicinus Nicot strain P-10. Glucose served as the major carbon source and nitrogen was supplied by five amino acids, L-arginine, L-aspartic acid, L-glutamic acid, glycine and DL-methionine. Omission of any of the first four diminished or prevented production of cephalosporin C; omission of methionine did not. Methionine is not critical for the production of cephalosporin C in this defined medium. Production of the antibiotic was affected by the concentrations of inorganic salts employed. Biotin was required for growth and cephalosporin C synthesis. The addition of L-lysine precursors to the medium did not influence cephalosporin C levels and L-lysine itself inhibited antibiotic production. Known precursors of fl-lactam antibiotics as well as oleic acid did not affect biosynthesis of cephalosporin C. Chemical changes occurring in the defined medium revealed that glucose was efficiently utilized after 96 hours incubation whereas total soluble nitrogen levels increased following an initial sharp decrease. Mycelial weight and cephalosporin C production were both maximal after 96 hours incubation. Mycelial nitrogen was highest after 48 hours incubation whereas mycelial lipid levels were greatest after 72 hours.