In this report, we demonstrate that a complex mammalian protein containing multiple disulfide bonds is successfully expressed in an E.coli-based cell-free protein synthesis system. Initially, disulfide-reducing activities in the cell extract prevented the formation of disulfide bonds. However, a simple pretreatment of the cell extract with iodoacetamide abolished the reducing activity. This extract was still active for protein synthesis even under oxidizing conditions. The use of a glutathione redox buffer coupled with the DsbC disulfide isomerase and pH optimization produced 40 μg/mL of active urokinase protease in a simple batch reaction. This result not only demonstrates efficient production of complex proteins, it also emphasizes the control and flexibility offered by the cell-free approach. © 2003 Wiley Periodicals, Inc.