In this study, we report the isolation and characterization of three novel hemolymph proteins that are believed to be involved in the innate immune response of horseshoe crabs, Tachypleus tridentatus. They include two closely related proteins, one that binds to the protein A of Staphylococcus aureus (PAP) and another that binds to the lipopolysaccharide of Escherichia coli (LBP). PAP binds specifically to staphylococcal protein A (SpA) with a K(D) of 3.86 x 10(-5) M, whereas LBP binds to lipopolysaccharide (LPS) with a K(D) of 1.03 x 10(-6) M. Both PAP and LBP are glycoproteins with an apparent molecular mass of about 40 kDa. N-terminal sequences of PAP and LBP showed 61.9 and 72.2% identity, respectively, to tachylectin-3, a lectin isolated from the amebocyte of T. tridentatus, previously characterized by its affinity to the O-antigen of LPS and blood group A antigen (Muta, T., and Iwanaga, S. (1996) Curr. Opin. Immunol. 8, 41-47). The third protein, a galactose-binding protein (GBP), was found to bind tightly to Sepharose CL-4B and could only be eluted from the column matrix with chaotropic agents, such as 4 M urea or 2 M guanidine hydrochloride. Further analysis indicated that GBP binds to D(+)-galactose with a K(D) of 2.47 x 10(-7) M. N-terminal sequence analysis showed that GBP shared a 50% identity with lectin L-6, identified in the granules of amebocyte of T. tridentatus. (Gokudan, S., Muta, T., Tsuda, R., Koori, K., Kawahara, T., Seki, N., Mizunoe, Y., Wai, S. N. , Iwanaga, S., and Kawabata, S. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 10086-10091). Lectin-L6 and tachylectin-3 are nonglycosylated intracellular proteins with about half the molecular mass of PAP, LBP, and GBP. GBP also binds to PAP and LBP with K(D) values of 1.25 x 10(-7) and 1.43 x 10(-8) M, respectively, and this binding is enhanced about 10-fold upon the addition of SpA and LPS to form the GBP.PAP.SpA and GBP.LBP.LPS complexes, respectively.