AbstractHuman promyelocytic cells (HL‐60) were labeled with35S‐sulfate and either3H‐glucosamine or3H‐serine as precursors. Accumulation of35S‐labeled macromolecules was approximately linear for up to 96 h, with a mean cell:medium ratio of 5.5:1, although activity/105viable cells reached a plateau level after 24 h. Virtually none of the cell‐associated proteoglycan was removed by trypsinization, consistent with a predominantly intracellular localization. Proteoglycan heterogeneity was investigated by DEAE‐Sephacel chromatography, isopyknic CsCI gradient centrifugation, and gel filtration chromatography. HL‐60 cells appeared to synthesize a single proteoglycan species, Kav = 0.46 on Sepharose CL‐4B and Kav = 0.32 on Sepharose CL‐6B, recovered primarily from the high‐density fractions of a dissociative CsCI gradient (ρ>1.40 g/l). Degradation products of lower charge density, lower buoyant density, and lower hydrodynamic size were also present, mainly in the cell pellets. The major proteoglycan was found to contain chondroitin sulfate chains of average Mr= 14.5 kD, yielding virtually 100% 4‐sulfated disaccharides on digestion with chondroitinase ABC. The proteoglycan was resistant to trypsin, chymotrypsin, plasmin, and papain, and the core protein Mr was approximately 20 kD by molecular sieve chromatography. Induction of HL‐60 cells with 0.15 dimethyl sulfoxide (DMSO) resulted in differentiation to a more mature granulocytic phenotype and was associated with a reduction in35S‐sulfate incorporation to 45% of control values or 32%, expressed as activity/105cells. Proteoglycans synthesized by DMSO‐treated cells were identical to those from untreated cells in terms of hydrodynamic size, glycosaminoglycan Mr, and sulfation.