Bone marrow-derived mast cells (BMMCs) contain chondroitin sulfate (CS)-E comprised of GlcA-GalNAc(4SO4) units and GlcA-GalNAc(4,6-SO4) units. GalNAc 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO4) residues of CS. On the basis of the specificity of GalNAc4S-6ST, it is thought that CS-E is synthesized in BMMC through the sequential sulfation by chondroitin 4-sulfotransferase (C4ST)-1 and GalNAc4S-6ST. In this paper, we investigated whether GalNAc4S-6ST and C4ST-1 are actually expressed in BMMCs in which CS-E is actively synthesized. As the bone marrow cells differentiate to BMMCs, level of C4ST-1 and GalNAc4S-6ST messages increased, whereas chondroitin 6-sulfotransferase (C6ST)-1 message decreased. In the extract of BMMCs, activity of GalNAc4S-6ST and C4ST but not C6ST were detected. The recombinant mouse GalNAc4S-6ST transferred sulfate to both nonreducing terminal and internal GalNAc(4SO4) residues; the activity toward nonreducing terminal GalNAc(4SO4) was increased with increasing pH. When CS-E synthesized by BMMCs was metabolically labeled with 35SO4 in the presence of bafilomycin A, chloroquine or NH4Cl, the proportion of the nonreducing terminal GalNAc(4,6-SO4) was increased compared with the control, suggesting that GalNAc4S-6ST in BMMC may elaborate CS-E in the intracellular compartment with relatively low pH where sulfation of the internal GalNAc(4SO4) by GalNAc4S-6ST preferentially occurs.