A fourth lectin (GS IV), a serologically Lewis b (Leb)-active binding lectin, was isolated from Griffonia simplicifolia seeds by affinity chromatography on an Leb (alpha-L-fucose-(1 leads to 2)-beta-D-Gal-(1 leads to 3)-[alpha-L-fucose-(1 leads to 4)]-beta-D-GlcNAc)-active fragment coupled to Synsorb, followed by elution with 0.1 N acetic acid. The purified lectin was shown to be homogeneous by polyacrylamide gel electrophoresis and analytical gel column chromatography on Sephadex G-200. A glycoprotein containing mannose, N-acetylglucosamine, fucose, and xylose (4:2:1:1), the lectin is a dimer composed of subunits of Mr = 29,000 and 27,000 with an aggregate Mr = 56,000. The GS IV lectin precipitate equally with blood group Leb fragment-bovine serum albumin (BSA) and H-active fragment (alpha-L-fucose-(1 leads to 2)-beta-D-Gal-(1 leads to 4)-[alpha-L-fucose-(1 leads to 3)]-beta-D-GlcNAc)-BSA conjugates but only poorly with a Lewis a fragment-BSA conjugate. Inhibition of precipitation with various mono- and oligosaccharides indicated that the lectin is specific for type 1 or 2 chains containing two alpha-L-fucosyl groups on C-2 of the D-galactosyl residue and C-3 or C-4 of the D-GlcNAc unit. One alpha-L-fucosyl residue on C-3 or C-4 of the D-GlcNAc residue of type 1 or 2 chains inhibited the reaction, although these oligosaccharides were less active than the oligosaccharides containing 2 alpha-L-fucosyl units. L-Fucose, methyl alpha- or beta-fucoside, and p-nitrophenyl alpha- or beta-L-fucoside did not inhibit the lectin. GSIV did not display mitogenic activity against mouse spleen cells or human peripheral lymphocytes.