Antibodies to the alpha and beta 2 subunits and site-directed antibodies that distinguish alpha subunits of the RI and RII subtypes have been used to study the biosynthesis and assembly of sodium channels. The RII sodium channel subtype is preferentially expressed in rat brain neurons in primary cell culture. Post-translational processing of alpha subunits includes incorporation of palmityl residues in thioester linkage and sulfate residues attached to oligosaccharides. The incorporation of [3H] palmitate into alpha subunits is inhibited by tunicamycin, indicating that it occurs in the early stages of biosynthesis but after co-translational glycosylation. Mature alpha subunits are attached to beta 2 subunits through disulfide bonds within 1 h after synthesis and up to 30% can be specifically immunoprecipitated from the cell surface with antibodies against the beta 2 subunits by 4 h after synthesis. The remaining alpha subunits remain in an intracellular pool. The alpha subunits synthesized in the presence of castanospermine and swainsonine have reduced apparent size. Castanospermine prevents incorporation of approximately 81% of the sialic acid of the alpha subunit and inhibits sulfation but not palmitylation. Although inhibition of glycosylation with tunicamycin blocks assembly of functional sodium channels, castanospermine and swainsonine do not prevent the covalent assembly of alpha and beta 2 subunits or the transport of alpha beta 2 complexes to the cell surface, and sodium channels synthesized under these conditions have normal affinity for saxitoxin. Thus, the extensive processing and terminal sialylation of oligosaccharide chains during maturation of the alpha subunit is not essential. A kinetic model for biosynthesis, processing, and assembly of sodium channel subunits is presented.