Temperature is a key environmental variable whose potential in animal cell fermentor optimization is not yet fully utilized. The scarce literature data suggests that reduced fermentor temperature results in an improved viability and shear resistance, higher cell density and titer in batch cultures, and reduction in glucose/lactate metabolism. Due to the arrest of the cells in the G1 phase, the specific growth rate was found to decrease at temperatures below 37.0°C. The response of the specific production rate was cell line dependent: in some cases it increased 2-to-3-fold, but decreased in other cases. The controlable slowdown of cell metabolism at lower temperature can be used in optimization of perfusion mammalian cell cultures with several potential advantages, including higher cell density in oxygen limited reactors, lower perfusion rate, improved product quality, simplified pH control, and others. To evaluate this strategy, a series of long-term experiments in 15 L perfusion bioreactors culturing recombinant hamster cells at 20.0 × 106 cells/mL were conducted. The temperature was changed over a range of set points, and maintained at each of these for a long period of time. Steady state process data was collected and analyzed. The effect of temperature on the following characteristics of the perfusion process was studied: cell growth, glucose/lactate metabolism, glutamine/ammonia metabolism, cell respiration, cell density at constant oxygen transfer rate, proteolytic activity, and product quality (glycosylation and molecule fragmentation). The results suggest that temperature is a variable with a significant potential in optimization of perfusion cultures. Properly selected temperature set point will contribute to the overall improvement of process performance. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng55: 328–338, 1997.