The fungus Cephalosporium acremonium produces two β-lactam antibiotics, penicillin N and cephalosporin C. These antibiotics are modified tripeptides derived from α-aminoadipic acid, cysteine, and valine. Mutant strains of C. acremonium fall into four phenotypic classes based on potential to synthesize antibiotics: (A) strains with increased potential to synthesize cephalosporin C, (B) strains unable to synthesize either antibiotic, (C) strains able to synthesize only penicillin N, and (D) strains able to synthesize penicillin N and 6-aminopenicillanic acid. Mutants of the second class, β-lactam negative mutants, have been characterized further. One mutant is auxotrophic for lysine and will synthesize antibiotic only if grown in media supplemented with α-aminoadipic acid or pipecolic acid. Other mutants are prototrophic and consistently negative for antibiotic synthesis. These inactive mutants are further subdivided according to their ability to form peptides implicated as intermediates for the synthesis of β-lactam antibiotics.