We evaluated the combined effect of decreasing the temperature to a mild hypothermia range (34 and 31°C) and switching to a slowly metabolizable carbon source (glucose substituted by galactose) on the growth and production of a recombinant human tissue plasminogen activator (rh-tPA) by Chinese hamster ovary cells in batch and semi-perfusion cultures. In batch cultures using glucose as a carbon source, decreasing the temperature caused a reduction in cell growth and an increase in specific productivity of rh-tPA of 32% at 34°C and 55% at 31°C, compared to cultures at 37°C. Similar behaviour was observed in cultures at 34°C using galactose as a carbon source. Nonetheless, at 31°C, the specific productivity of rh-tPA strongly decreased (about 58%) compared to the culture at 37°C. In semi-perfusion culture, the highest rh-tPA specific productivity was obtained at 34°C. Similarly, whether a decrease in the temperature is accompanied of the replacement of glucose by galactose, the rh-tPA specific productivity improved about 112% over that obtained in semi-perfusion culture carried out at 37°C with glucose as the carbon source. A semi-perfusion culture strategy was implemented based on the combined effect of the chosen carbon source and low temperatures, which was a useful approach for enhance the specific productivity of the recombinant protein.