The P-selectin counterreceptor PSGL-1 is covalently modified by mono alpha2,3-sialylated, multiply alpha1,3-fucosylated polylactosamines. These glycans are required for the adhesive interactions that allow this adhesion receptor-counterreceptor pair to facilitate leukocyte extravasation. To begin to understand the biosynthesis of these glycans, we have characterized the acceptor and site specificities of the two granulocyte alpha1,3-fucosyltransferases, Fuc-TIV and Fuc-TVII, using recombinant forms of these two enzymes and a panel of synthetic polylactosamine-based acceptors. We find that Fuc-TIV can transfer fucose effectively to all N-acetyllactosamine (LN) units in neutral polylactosamines, and to the "inner" LN units of alpha2,3-sialylated acceptors but is ineffective in transfer to the distal alpha2,3-sialylated LN unit in alpha2,3-sialylated acceptors. Fuc-TVII, by contrast, effectively fucosylates only the distal alpha2,3-sialylated LN unit in alpha2,3-sialylated acceptors and thus exhibits an acceptor site-specificity that is complementary to Fuc-TIV. Furthermore, the consecutive action of Fuc-TIV and Fuc-TVII, in vitro, can convert the long chain sialoglycan SAalpha2-3'LNbeta1-3'LNbeta1-3'LN (where SA is sialic acid) into the trifucosylated molecule SAalpha2-3'Lexbeta1-3'Lexbeta1-3'Lex (where Lex is the trisaccharide Galbeta1-4(Fucalpha1-3)GlcNAc) known to decorate PSGL-1. The complementary in vitro acceptor site-specificities of Fuc-TIV and Fuc-TVII imply that these enzymes cooperate in vivo in the biosynthesis of monosialylated, multifucosylated polylactosamine components of selectin counterreceptors on human leukocytes.