Nonribosomal enterobactin synthetase of Escherichia coli was found to prematurely release a large amount of linear precursors in an in vitro reconstitution. However, these side products are suppressed to negligible levels by polymeric cosolvents that create macromolecular crowding, a prominent feature of the intracellular environment. These findings show that macromolecular crowding is essential to normal functioning of the nonribosomal peptide synthetase and suggest that it may be crucial to biotechnological utilization of similar enzyme systems.