Glycosaminoglycans (GAGs) are polyanionic components of the cell surface that have been shown to play an important role in the cellular differentiation of many embryonic systems, as well as in the maturation of the developing human leukocyte. For this reason, the production of GAGs during the induction of myelocytic and macrophage-like differentiation of the human promyelocytic leukemia cloned cell line HL60/HGPRT- was studied. The major GAG component of HL60/HGPRT- was chondroitin 4-sulfate. This molecule has been reported to be the major GAG constituent of normal granulocytes and myeloid leukemia cells as well. Treatment of HL60/HGPRT- cultures with dimethyl sulfoxide, which initiates myeloid maturation, or 12-O-tetradecanoylphorbol-13- acetate, which induces the formation of macrophage-like cells, resulted in a 43 and 34% reduction, respectively, of the incorporation of [35S]sulfate into total GAGs at a time when greater than 80% of the cells were morphologically immature and were unable to reduce nitroblue tetrazolium dye. This reduction occurred primarily in GAGs associated with the cells, which decreased by 75% after exposure to these agents. Therefore, the distribution of GAGs between the cellular and medium compartments was altered by exposure to inducers. A phorbol ester with no capacity to induce differentiation, 4 alpha-phorbol-12, 13-didecanoate, elicited a reproducible but less dramatic decrease in cell-associated GAGs. The reduction in [35S]-sulfate incorporation into GAGs, therefore, may be an important step in leukocyte differentiation and may provide a useful biochemical probe of the maturation process.