Stable transformation was used to generate a cloned insect cell line (Bm5 silkmoth cells) overexpressing human tissue plasminogen activator (tPA). This cell line expressed 135 mu g/mL single chain tPA in serum-free medium in static culture with a maximum specific activity of 120 IU/mu g. In serum-containing medium, this line expressed 160 mu g/mL of combined single-chain tPA, two-chain tPA, and a higher molecular weight SDS-stable tPA complex in suspension cultures with a maximum specific activity of 255 IU/mu g. Approximately 100 copies of the tPA cDNA were randomly integrated into each Bm5 cell. For secretion of recombinant tPA from Bm5 cells, the native human tPA signal peptide is as effectively recognized as an insect specific signal peptide derived from a silkmoth chorion gene. Finally, stably transformed polyclonal populations of Bm5, High Five, and Sf21 cells expressing tPA were generated and compared for relative tPA expression.