Previously, we have reported that purification and characterization of an anti-plant viral serine protease (EF-SP1) from Eisenia foetida. In this study, a new serine protease (EF-SP2) showing strong antiviral activities against cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) was purified from the coelomic fluid of the earthworm E. foetida. The activity of EF-SP2 was suppressed by various known serine protease inhibitors, suggesting that the EF-SP2 is a serine protease. Its molecular weight was estimated to be 26,000 by SDS-PAGE, and its optimal pH and temperature were pH 9.5 and 60 C, respectively. N-terminal amino acid sequence of EF-SP2 was the same as those of E. foetida serine proteases (EFE-d and EFE-e) with fibrinolytic activity, but different from that of EF-SP1. The enzymatic properties of anti-plant serine proteases (EF-SP1 and EF-SP2) and fibrinolytic enzymes (EFE-d and EFE-e) were similar to each other, e.g., substrate specificity, molecular weight, and effect of inhibitors. Our results suggest that EF-SP2 as well as EF-SP1 can be also applicable as a potential antiviral factor against CMV, TMV, and other plant viruses. (C) 2011 Elsevier Ltd. All rights reserved.