The effect of culture temperature on urokinase production by HT-1080 cell line was studied in batch culture and hollow fiber reactor. Small-scale t-flask experiments revealed that urokinase production could be enhanced and media utilization could be reduced by lowering the culture temperature in production phase. Urokinase production was scaled up using a hollow fiber perfusion reactor system. Temperature of culture was maintained at the physiological 37 A degrees C during growth phase that extended up to 12 days in hollow fiber bioreactor. Subsequently, in the production phase, culture temperature was lowered to 34 A degrees C. Decrease in culture temperature resulted in a significant increase in urokinase production. Proteolytic degradation and inhibition was also minimized. The medium utilization rate was decreased at lower temperature, and hence, a higher economy of production could be obtained.