Objective To use a transient expression system to express a truncated human tissue plasminogen activator (K2S) gene in cucurbit plants. Results The recombinant tissue plasminogen activator protein (K2S form) was expressed in active form in cucurbit plants. Its molecular weight was 43 kDa. The plant-derived rt-PA was determined using goat anti-rabbit antibody by western blotting. Among the infected lines, the highest expression of rt-PA was 62 ng/100 mg per leaf tissue as measured by ELISA. The enzymatic activity of the plant-derived rt-PA was 0.8 IU/ml. Conclusions The K25 form of rt-PA was expressed for the first time using the viral expression system. Plant-derived rt-PA showed similar potency to commercially-available PA.