A baby hamster kidney (BHK) cell line producing recombinant tissue plasminogen activator (tPA) was cultured with control of both the glucose and glutamine concentrations by an on-line HPLC measuring and control system. It was found that BHK cell growth showed more significant responses to glutamine than to glucose. An approximately 2-fold increase in the viable cell concentration was observed when glutamine was controlled at the low levels of 0.1 and 0.5 g/l compared to that when it was controlled at 1.0 g/l. Maximum tPA production was obtained in a control culture with 1.0 g/l-glucose and 0.5 g/l-glutamine. Accumulation of lactate and ammonia showed respectively less dependence on the glucose and glutamine concentrations in the medium.