Metabolites (both intra- and extracellular) involved in penicillin biosynthesis were measured during fed-batch cultivations with a high-yielding strain of Penicillium chrysogenum. The fed-batch cultivations were carried out on a complex medium containing corn steep liquor. Three distinct phases were observed: (a) a rapid growth phase where free amino acids present in the medium are metabolized, (b) a linear growth phase, and (c) a stationary phase. The specific penicillin production (rp) is initially high and, during the rapid growth phase, it increases slightly. During the linear growth phase rp is approximately constant [4-6 mg penicillin V (g dry weight)-1 h-1 depending on the operating conditions], whereas it decreases during the stationary phase. During the cultivations the tripeptide Aad-Cys-Val (the first metabolite in penicillin biosynthesis) and 8-hydroxypenillic acid (formed by carboxylation of 6-aminopenicillanic acid, 6-APA) were found to accumulate in the medium, whereas the concentrations of isopenicillin N and 6-APA were found to be approximately constant and low. About 3% of the Aad-Cys-Val formed in the first step of the penicillin biosynthetic pathway is lost to the medium and 4% of the isopenicillin N formed in the second step of the pathway is lost as extracellular isopenicillin N, 6-APA or 8-hydroxypenillic acid. Also the cyclic form of alpha-aminoadipic acid, 6-oxo-piperidine-2-carboxylic acid, was found to accumulate in the medium and it was found to be formed in an approximately constant ratio to penicillin V of 6 mol/100 mol.