The human promyelocytic leukemia ell line, HL-60, synthesized a class of high-molecular-weight (M.W. 5000 to 7000), N-linked glycopeptides as the major class of protein-bound carbohydrates. Small glycopeptides (M.W. 2500 to 3500), typical of most mammalian cells except erythrocytes, represented a minor component in these cells. The large glycopeptides were labeled efficiently with fucose, glucosamine, and galactose but only poorly with mannose. They were found not to be glycolipids, glycosaminoglycans, or mucin-type glycopeptides and were not susceptible to exoglycosidases, but they were partially degraded by endo-beta-galactosidases. These characteristics are similar to those of the large glycopeptides synthesized by erythrocytes, by another human myeloid leukemia cell line (K562), and by human and murine teratocarcinoma cells. High-molecular-weight glycopeptides predominated on another human myeloid leukemia cell line KG1, but they were expressed at low levels on both a human monocytic leukemia cel line (THP-1) and a human T-lymphoblastoid cell line (Jurkat). When HL-60 cells were induced to differentiate into macrophage-like cells with phorbol esters, the proportion of large glycopeptides decreased, and the production of small glycopeptides predominated. This shift was observed within the first several hr after exposure to phorbol esters and was temporally related to the acquisition of adherent properties by the induced cells. In contrast, when HL-60 cells were induced to differentiate into granulocytes by dimethyl sulfoxide, hypoxanthine, or retinoic acid, they continued to synthesize glycopeptides similar to uninduced cells. Human peripheral blood granulocytes synthesized primarily large glycopeptides, whereas monocytes and lymphocytes synthesized mostly small glycopeptides. These results indicate that the synthesis of high-molecular-weight glycopeptides is a property of human myeloid leukemia cell lines and that it persists throughout myeloid differentiation. A proportionate decrease in the synthesis of these large glycopeptidase is a part of the differentiation program for monocytes and macrophages.