Neurofilaments, the major intermediate filaments in large myelinated neurons, are essential for specifying proper axonal caliber. Mammalian neurofilaments are obligate heteropolymers assembled from three polypeptides, neurofilament (NF)-H, NF-M, and NF-L, each of which undergoes phosphorylation at multiple sites. NF-M and NF-L are known to be modified by O-linked N-acetylglucosamine (O-GlcNAc) (Dong, D. L.-Y., Xu, Z.-S., Chevrier, M. R., Cotter, R. J., Cleveland, D. W., and Hart, G. W. (1993) J. Biol. Chem. 268, 16679-16687). Here we further report that NF-H is extensively modified by O-GlcNAc at Thr53, Ser54, and Ser56 in the head domain and, somewhat surprisingly, at multiple sites within the Lys-Ser-Pro repeat motif in the tail domain, a region in assembled neurofilaments known to be nearly stoichiometrically phosphorylated on each of the approximately 50 KSP repeats. Beyond the earlier identified sites on NF-M and NF-L, O-GlcNAc sites on Thr19 and Ser34 of NF-M and Ser34 and Ser48 of NF-L are also determined here, all of which are localized in head domain sequences critical for filament assembly. The proximity of O-GlcNAc and phosphorylation sites in both head and tail domains of each subunit indicates that these modifications may influence one another and play a role in filament assembly and network formation.