Based on assumed reaction network structures, NADPH availability has been proposed to be a key constraint in β-lactam production by Penicillium chrysogenum. In this study, NADPH metabolism was investigated in glucose-limited chemostat cultures of an industrial P. chrysogenum strain. Enzyme assays confirmed the NADP+-specificity of the dehydrogenases of the pentose-phosphate pathway and the presence of NADP+-dependent isocitrate dehydrogenase. Pyruvate decarboxylase/NADP+-linked acetaldehyde dehydrogenase and NADP+-linked glyceraldehyde-3- phosphate dehydrogenase were not detected. Although the NADPH requirement of penicillin-G-producing chemostat cultures was calculated to be 1.4-1.6-fold higher than that of non-producing cultures, in vitro measured activities of the major NADPH-providing enzymes were the same. Isolated mitochondria showed high rates of antimycin A-sensitive respiration of NADPH, thus indicating the presence of a mitochondrial NADPH dehydrogenase that oxidises cytosolic NADPH. The presence of this enzyme in P. chrysogenum might have important implications for stoichiometric modelling of central carbon metabolism and β-lactam production and may provide an interesting target for metabolic engineering.