Aiming at maximizing the production of transmembrane rabies virus glycoprotein (rRVGP), the influence of hypothermic temperature on a recombinant Drosophila melanogaster S2 cell culture in Sf-900II medium was investigated. Cell growth and rRVGP production were assessed at 4 culture temperatures in Schott flasks: 16, 20, 24 and 28°C. The maximum specific growth rates μ max were, respectively: 0.009, 0.019, 0.038 and 0.035h −1 , while the maximum rRVGP levels CmaxrRVGP were: 0.075, 2.973, 0.480 and 1.404mgL −1 . The best production temperature (20°C) was then tested in a bioreactor with control of pH and dissolved oxygen in batch and fed-batch modes. In the batch culture, μ max and CmaxrRVGP were 0.060h −1 and 0.149mgL −1 at 28°C and 0.026h −1 and 0.354mgL −1 at 20°C, respectively. One batch-culture experiment was carried out with adaptation of the cells by the temperature falling in steps from 20°C to 16°C, so that μ max fell from 0.023 to 0.013h −1 , while CmaxrRVGP was improved to 0.567mgL −1 . In the fed-batch mode at 20°C, μ max was 0.025h −1 and CmaxrRVGP was 1.155mgL −1 . Taken together, these results indicate that the best strategy for optimized rRVGP production is the culture at hypothermic temperature of 20°C, when μ max is kept low and with feeding of limitant aminoacids.