A high-concentration medium was developed for the high-density cultivation of Chinese hamster ovary (CHO) cells to produce tissue plasminogen activator (WA). First, the effects of the concentrations of several medium components on the growth and metabolism of CHO cells were studied particularly in relation to the serum concentration, in order to develop a suitable concentrated medium. High concentrations of serum, glucose, lactate, and glutamine and a low ammonia concentration increased the specific growth rate and decreased the specific tPA production rate. Among these components, the effect of the serum concentration was the most significant. There was an inverse correlation between the specific rates of cell growth and tPA production. Using a medium containing glutamine at twice the concentration of the original medium (F-12) allowed the medium volume to be reduced to seven-ninths that of the original, without any reduction in tPA productivity. When the F-12 medium was supplemented five-fold with amino acids and vitamins, the total amount of tPA produced, specific rate of tPA production, and yield of tPA per unit volume of medium employed were respectively enhanced 1.7, 1.5, and 3.6 times. As a consequence, the frequency of medium exchange could be reduced. The results strongly indicate that the supplementation of nutrient amino acids and vitamins to a medium with a low concentration of serum are the most critical factors for cost-effective tPA production in high-density cell cultivation.