The influence the of dissolved oxygen concentration on penicillin biosynthesis was studied in steady-state continuous cultures of a high-yielding strain of Penicillium chrysogenum operated at a dilution rate of 0.05 h-l. The dissolved oxygen concentration was varied between 0.019 and 0.344 mM (corresponding to 7% and 131% air saturation at 1 bar) solely through manipulations of the inlet gas composition. At dissolved oxygen concentrations above 0.06-0.08 mM, a constant specific penicillin productivity of around 22 (mu mol/g of DW)/h is maintained. At lower oxygen concentrations, the specific penicillin productivity decreases, and a value of 17 (mu mol/g of DW)/h was obtained when the dissolved oxygen concentration was 0.042 mM. A further lowering of the dissolved oxygen concentration to 0.019 mM resulted in the loss of penicillin production. However, penicillin productivity was instantly recovered to its maximum value when the dissolved oxygen concentration was reset to a value above 0.08 mM. The specific formation rates of a number of typical byproducts of the penicillin production, i.e., delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, isopenicillin N, 6-aminopenicillanic acid, 8-hydroxypenillic acid and 6-oxopiperidine-2-carboxylic acid, the delta-lactam form of cl-aminoadipic acid, all increased with decreasing dissolved oxygen concentration. A simultaneous increase in the secretion of glutathione was observed, and a link between the secretion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and glutathione is suggested. The intracellular pools of the pathway intermediates delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N increased respectively 2- and 3-fold when the dissolved oxygen concentration was lowered from 0.344 to 0.042 mM, whereas the intracellular pools of glutathione and cysteine decreased at low dissolved oxygen concentrations. On the basis of the intracellular pool measurements, metabolic control analysis is performed, and the flux control coefficients for the first two enzymes in the penicillin biosynthetic pathway, i.e., delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase and isopenicillin N synthetase, are calculated at different dissolved oxygen concentrations. It is found that for low dissolved oxygen concentrations, the flux control is mainly exerted by the isopenicillin N synthetase, whereas a more even distribution of the flux control by the two enzymes is encountered at high dissolved oxygen concentrations.