A recombinant DNA Chinese hamster ovary (CHO) cell line which produces tissue-type plasminogen activator (t-PA) was cultivated continuously in suspension with a constant dilution rate of 0.5 day−1. The cultivation consisted of four phases with four different ammonium chloride concentrations (0,2.5, 5, and 7.5 mM) in the feed medium, causing a reactor ammonium concentration of up to 8 mM. Cell growth was not inhibited by these high ammonium concentrations, as cell densities of around 2.3 × 106 cells mL−1 were established. In contrast, the production of t-PA was reduced under high ammonium concentration. The decrease in specific t-PA production could be due to either a negative ammonium influence on productivity or a limitation of medium components, e.g., amino acids. Cell metabolism was changed under high ammonium concentrations, seen most clearly by a decrease in specific ammonium production by a factor of 8 and an increase in specific alanine production of 30%.