A stoichiometric equation for the biosynthesis of actinorhodin (ACT) was derived taking into consideration both the requirements of the carbon precursors (acety1-CoA) and reducing power (NADPH). The estimate for reducing power was derived from a detailed molecular analysis of each step in the ACT biosynthetic pathway. Even though ACT is slightly more oxidized than most carbon substrates, e.g. glucose, reducing power (NADPH and NADH) is necessary due to reducing steps and to monooxygenase steps. The equation was used to evaluate, in a metabolic network context, the experimental results from batch fermentations with eight different carbon sources using a Streptomyces lividans 1326 derived strain containing the pat way-specific activator gene (actII-ORF4) on a multicopy plasmid (pIJ68). The yield of ACT on the various carbon sources ranged from 0.04 to 0.18 Cmol ACT/Cmol carbon source in the stationary phase. Glucose was the best carbon source and supported a yield of 25% of the maximum theoretical yield. There are no obvious constraints in the primary metabolic pathways that can explain why the various carbon sources allowed different levels of ACT production, because their potential for supplying acety1-CoA and NADPH are far from fully utilized. For the observed ACT yields, there is an excess production of NADPH that has to be reoxidized either by a transhydrogenase or a NADPH oxidase. This study discusses the central metabolic pathways, focusing on providing precursors for ACT synthesis.