Glycosaminoglycans (GAGs) play an important role in cell-cell and cell-substratum interactions, and undergo specific changes during neutrophil development. Previous studies (Luikart, S.D., Maniglia, C. A., and Sartorelli, A. C. Cancer Res., 44: 2907-2912, 1984) have shown that both dimethyl sulfoxide and 4-beta-phorbol-12-beta-myristate-13-alpha-acetate decreased GAG production by a hypoxanthine-guanine phosphoribosyl transferase-deficient clone of HL-60 promyelocytic leukemia cells prior to the appearance of a mature myeloid or monocytoid phenotype. To expand these investigations further, GAGs were analyzed by cetylpyridinium chloride precipitation and DEAE-Sephacel ion-exchange chromatography after labeling of parental HL-60 cultures with [35S]sulfate and D-[3H]glucosamine for 6 h, following treatment with 1 microM all-trans retinoic acid (RA). Chondroitin sulfate represented the major GAG species produced, although endo-beta-galactosidase-sensitive undersulfated macromolecules which possibly might be keratan sulfate, were also identified. GAG production decreased over a time period of 144 h in culture. RA treatment reduced the amount of radiolabeled cell-associated GAGs by 50% after 48, 96, and 144 h of exposure. In contrast, commitment to myelocytic maturation of the majority (i.e., approximately 60%) of the cells occurred between 72 and 96 h of RA treatment. Concurrently with the appearance of mature granulocytic cells, two-thirds of the radiolabeled GAGs were recovered from the medium, compared to one-third in untreated cultures, a phenomenon that resulted in an overall alteration in the distribution of GAGs. When RA was removed by washing after either 48 h (i.e., precommitment to differentiation) or 96 h (i.e., postcommitment to differentiation), a 1.5- to 3.5-fold increase in GAG production was noted 48 h later; this increase was unrelated to the medium change or to alterations in cell cycle distribution. The amounts of endo-beta-galactosidase-sensitive macromolecules were unaltered. Thus, although 1 microM RA inhibited the synthesis of chondroitin sulfate by HL-60 leukemia cells, this inhibition was reversible by removal of the drug and appeared to be unrelated to the commitment to myelocytic maturation.