The efficient production of tissue plasminogen activator by human diploid fibroblasts is described. Confluent fibroblasts lost their tissue plasminogen activator-producing ability when incubated in a serum-free control medium containing proteose peptone (10 mg ml(-1)) for longer than 4 to 7 d. In comparison, the cells continued producing the enzyme for more than 14 d when incubated in an experimental medium containing yeast mannan (2 mg ml(-1)) and CaCl2 (3.6 mM) in addition to peptone. The productivity of the enzyme maximally increased when the ratio of the medium volume to the monolayer area decreased. Such an improved culture system yielded 29 times the productivity achieved under the control conditions. The use of mannan is practically important for large-scale production and subsequent purification of the enzyme.