Hepatocyte growth factor, also known as scatter factor, is a complete mitogen for hepatocytes that bears sequence and structural homology with plasminogen. Because it exists in both a mitogenically inactive single-chain form and an active two-chain form, we were interested in determining whether plasminogen activators could properly cleave single-chain hepatocyte growth factor to generate active two-chain hepatocyte growth factor. Herein we report that both urokinase-type plasminogen activator and tissue-type plasminogen activator can cleave single-chain hepatocyte growth factor, generating two-chain hepatocyte growth factor. When equal quantities of plasminogen activator-treated and activator-untreated hepatocyte growth factor are compared in serum-free in vitro bioassays, the treated hepatocyte growth factor is mitotically more active. Also, urokinase-type plasminogen activator was inactive against hepatocyte growth factor molecules with a mutated cleavage site. This suggests that urokinase-type and tissue-type plasminogen activator may be natural biological regulators of hepatocyte growth factor. Because the active form of hepatocyte growth factor is a powerful stimulator of DNA synthesis and cell motility, these findings may be relevant in understanding the role of plasminogen activators in the biology of cancer invasion and metastasis.