The T cell and NK cell adhesion molecule CD2 interacts with different ligands, viz, CD58, CD48, and CD59. Using a fluorescent multimeric construct of rCD2, we previously identified an additional CD2 ligand (CD2L) on the erythroleukemic cell line K562. CD2L bound to a different region of CD2 than known ligands and was N-glycosylation dependent. In this study we show that mAbs specific for the carbohydrate Ag Lewis x (CD15, Gal-beta 1-4 GlcNAc alpha 1-3Fuc) inhibit multimeric rCD2 binding to CD2L. CD2L is restricted in expression to myeloid cells, where it is co-expressed with CD58 on monocytes and is the dominant, if not sole, CD2 ligand on neutrophils. Sugar specificity studies show that CD2L is not CD15. Thus, whereas soluble Lewis x inhibits binding of CD15 mAb to K562 and neutrophils, binding of multimeric rCD2 is unaffected. Furthermore, multimeric rCD2 binding to K562 is inhibited by L-fucose and following treatment of K562 with an alpha 1-6 fucosidase, whereas these treatments do not inhibit the binding of CD15 mAb. Thus, it is likely that CD2L is a carbohydrate structure closely associated with, yet distinct from, CD15, which can be sterically blocked by CD15 mAb. Functional studies revealed that CD2L is probably an important CD2 ligand in the non-MHC-restricted NK cell killing of K562 target cells, since this activity was strongly inhibited by CD15 mAb. Collectively, this study indicates that a CD15 (Lewis x)-associated carbohydrate structure(s), which has previously been shown to be a selectin ligand, also may function as an important CD2 ligand on myeloid cells.