Based on the effects of temperature shift on the cell cycle, apoptosis and metabolism of a recombinant Chinese hamster ovary (rCHO) cell line (CL-11G) producing pro-urokinase (pro-UK) in batch cultures, the potential of temperature shift as a tool in the optimization of the perfusion culture of CL-11G cells for the production of pro-UK was examined. The proportion of CL-11G cells in the G(0)/G(1) phase in static cultures increased from 56.4% to 82.8% following a temperature shift from 37degreesC to 31degreesC. Conversely, the proportion of CL-11G cells in the S phase decreased from 34.8% to 11.6%. The specific growth rate of CL-11G cells reflected the effect of temperature on the cell cycle and decreased from 0.024 h(-1) at 37degreesC to 0.006 h(-1) at 31degreesC. Continuous exposure to the non-permissive temperature of 31degreesC led to a marginal increase in apoptosis. The specific pro-UK productivity of CL-11G cells increased by 74% at 34degreesC compared with controls at 37degreesC in batch cultures. CL-11G cells immobilized with Cytopore 1 in a 5-l bioreactor initiated at 37degreesC and temperature shifted to WC exhibited an average 17% increase in viable cell density and an average 47% increase in pro-UK production. These results demonstrated that temperature shift offers the prospect of enhancing the productivity of pro-UK in high-density perfusion culture.