Based on a review of the Penicillium chrysogenum biochemistry a stoichiometric model has been set up. The model considers 61 internal fluxes and there are 49 intracellular metabolites which are assumed to be in pseudo-steady state. In addition to the intracellular fluxes the model considers the uptake of 21 amino acids. From the stoichiometric model the maximum theoretical yield of penicillin V is calculated to 0.43 mol/mol glucose. If biosynthesis of cysteine is by direct sulfhydrylation rather than by transsulfuration, the maximum theoretical yield is about 20% higher, i.e., 0.50 mol/mol glucose. The theoretical yield decreases substantially if alpha-aminoadipate is converted to 6-oxo-piperidine-2-carboxylic acid (OPC). If only 40% of the alpha-aminoadipate is recycled, the maximum theoretical yield is 0.31 mol/mol glucose. The uptake rates of glucose, lactate, gamma-aminobutyrate, and 21 amino acids were measured during fed-batch cultivations. The rates of formation of penicillin V, delta-(L-alpha-aminoadipyl-L-cysteinyl-D-valine (ACV), OPC, and the pool of isopenicillin N, 6-APA, and 8-HPA were also measured. Finally the synthesis rates of the biomass constituents RNA/DNA, protein, lipid, carbohydrate, and amino carbohydrate were measured. From these measured rates and the stoichiometric model the metabolic fluxes through the different intracellular pathways are calculated. The calculations show that penicillin formation is accompanied by a large flux through the pentose phosphate (PP) pathway due to a large requirement for nicotinamide-adenine dinucleotide phosphate (NADPH) used in the biosynthesis of cysteine. If cysteine is added to the medium, the flux through the PP pathway decreases. From the stoichiometric model Y-xATP is calculated to 87 mmol adenosine triphosphate (ATP)/g dry weight (DW), and from the flux calculations m(ATP) is found to 3 mmol ATP/g DW/h.