Vibrio mimicus (VM) causes both intestinal and extraintestinal infections. A proteolytic hemagglutinin (HA) has been described for VM and is a metallo-protease which only agglutinates 1.5% chicken erythrocytes (RBCs). In this study, a surface protein identified by HA activity with 0.3% sheep RBCs and possessing proteolytic activity was isolated and characterized. Using RBC concentrations from 0.3 to 1.5 %, the HA agglutinated sheep, chicken, bovine, rabbit, guinea pig, and human (A, O, and B) RBCs. This protein has a molecular weight of 30 and 33 Kdal by gel filtration and SDS-PAGE, respectively and has isoelectric points of 5.15, 5.2, 5.55, and 5.6. Electron microscopy of samples revealed aggregated globular proteins of various stoichiometry. Protease activity was inhibited by orthophenanthroline (OPA), Zincov (ZN), and Phosphoramidon. However, HA activity was inhibited only by OPA and ZN. Comparison of the N-terminal amino acid (AA) sequence of this protein with that previously reported demonstrated a single AA difference (Asn to Val) in position number 2. These data suggest that the VM protease has a much broader HA species spectrum than previously reported and that the active sites for HA and proteolytic activities may be on different parts of the molecule.