This paper describes the thermodynamic analysis of pathways related to penicillin production in Penicilliumchrysogenum. First a thermodynamic feasibility analysis is performed of the l-lysine pathway of which one of the precursors for penicillin biosynthesis (α-aminoadipic acid) is an intermediate. It is found that the l-lysine pathway in P. chrysogenum is thermodynamically feasible and that the calculated standard Gibbs free energy values of the two enzymes controlling the pathway flux indicate that they operate far from equilibrium. It is therefore proposed that the regulation of α-aminoadipate reductase by lysine is important to maintain a high concentration of α-aminoadipate in order to direct the carbon flux to penicillin production. Secondly the changes in Gibbs free energy in the penicillin biosynthetic pathway during fed-batch cultivation were studied. The analysis showed that all reactions in the pathway were thermodynamically irreversible at physiological conditions during fed-batch cultivations. Furthermore it was observed that the higher thermodynamic driving force of IAT could explain why penicillin is mainly formed from isopenicillin N in a one-step reaction, as it has been previously proposed.