Three popular expression host systems Escherichia coli, Pichia pastoris and Drosophila S2 were analyzed techno-economically using HIV-1 Nef protein as the model product. On scale of 100 mg protein, the labor costs corresponded to 52-83% of the manufacturing costs. When analyzing the cost impact of the different phases (strain/cell line construction, bioreactor production, and primary purification), we found that with the microbial host systems the strain construction phase was most significant generating 56% (E. coli) and 72% (P. pastoris) of the manufacturing costs, whereas with the Drosophila S2 system the cell line construction and bioreactor production phases were equally significant (46 and 47% of the total costs, respectively). With different titers and production goal of 100 mg of Nef protein, the costs of P. pastoris and Drosophila S2 systems were about two and four times higher than the respective costs of the E. coli system. When equal titers and bioreactor working volumes (10 L) were assumed for all three systems, the manufacturing costs of the bioreactor production of the P. pastoris and Drosophila S2 systems were about two and 2.5 times higher than the respective costs of the E. coli system. 0 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 95-102, 2009