Perfusion cultures of CHO cells producing t-PA were performed using acoustic filter cell retention. A robust off-line glucose analysis and predictive control protocol was developed to maintain the process within approximately 0.5 mM of the glucose set point, without the need for a more fallible on-line sensor. Glucose usage (the difference between the inlet and reactor glucose concentrations) provided an easily measured indicator of overall medium utilization for mapping acceptable ranges of operation, including the edge of failure. Earlier onset of perfusion with a ramping glucose set point (1.5 mM/d) resulted in improved growth and consistency during the perfusion culture start-up. At steady state, the t-PA concentration variability increased gradually with increasing glucose usage up to approximately 22 mM, then up to 24 mM the variability increased threefold. Peak t-PA concentrations of over 90 mg/L were obtained by controlling at a glucose usage of approximately 24 mM, but these t-PA levels were not sustainable for more than 3 days. A consistent t-PA concentration of 40 mg/L was obtained at a glucose usage of 21.5 mM. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 252–256, 2001.