Urokinase was produced in a hollow fiber reactor using HT-1080 human fibrosarcoma cells. External modulation comprised replenishing of the medium in the extracapillary space, reducing the serum concentration in the extracapillary space from 10% to 2% and increasing flow rate of the circulating medium in the intracapillary space from 20 to 80 mL/min, each according to a specific protocol. More than sixfold increase was observed in the cumulative urokinase production for two and three medium replenishing modulations of the extracapillary space. After 15 days of continuous operation, the highest cumulative urokinase obtained was 1.63 x 10(6) PU/mL. SDS-PAGE and zymogram study established that the urokinase obtained was in the high molecular weight range of 54 kDa. The effect of external modulation on cumulative urokinase production was visualized as trajectories with respect to the ratio of lactic acid production rate (LPR) to the glucose uptake rate (GUR). The collective external modulation data showed two separate physiological regions in the cumulative urokinase vs. LPR/GUR plane. The HT-1080 cells exhibited two distinct morphologies in these regions that may be related to acidosis and metastasis. These regions also correspond to low and high urokinase productivity.