A key determinant of influenza virus pathogenesis is mutation in the proteolytic cleavage site of the hemagglutinin (HA). Typically, low-pathogenicity forms of influenza virus are cleaved by trypsin-like proteases, whereas highly pathogenic forms are cleaved by different proteases (e.g., furin). Influenza virus A/WSN/33 (WSN) is a well-studied H1N1 strain that is trypsin independent in vitro and has the ability to replicate in mouse brain. Previous studies have indicated that mutations in the neuraminidase (NA) gene allow the recruitment of an alternate protease (plasminogen/plasmin) for HA activation. In this study we have identified an additional mutation in the P2 position of the WSN HA cleavage site (S328Y) that appears to control virus spread in a plasmin-dependent manner. We reconstructed recombinant WSN viruses containing tyrosine (Y), phenylalanine (F), or serine (S) in the P2 position of the cleavage site. The Y328 and F328 viruses allowed plaque formation in the absence of trypsin, whereas the S328 virus was unable to form plaques under these conditions. In mice, Y328 and F328 viruses were able to efficiently spread following intracranial inoculation; in contrast, the S328 virus showed only limited infection of mouse brain. Following intranasal inoculation, all viruses could replicate efficiently, but with Y328 and F328 viruses showing a limited growth defect. We also show that wild-type HA (Y328) was more efficiently cleaved by plasmin than S328 HA. Our studies form the foundation for a more complete understanding of the molecular determinants of influenza virus pathogenesis and the role of the plasminogen/plasmin system in activating HA.