Hepatocyte growth factor is a plasminogen-like molecule with diverse biological effects. Although it is synthesized as a single chain polypeptide, it was originally purified as a disulfide-linked heterodimer which was generated by an internal proteolytic event. Subsequent work indicated that preparations consisting largely of the monomeric form also exhibited potent activity. By using a combination of protease inhibition and site-directed mutagenesis, we established that conversion of the single chain polypeptide to the heterodimer occurred during the bioassay and was required for mitogenic and motogenic activity.