Monoclonal antibody 5B4 recognizes a carboxy-terminal epitope common to large (approximately 180 kDa) and short (approximately 140 kDa) forms of neural cell adhesion molecules (NCAM-ld and NCAM-sd, respectively). The deduced primary sequence of rat brain 5B4/NCAM-ld predicts a large cytoplasmic domain (390 amino acids, Mr 39,284) of striking amino acid composition (52% proline, alanine, serine and threonine) and little predicted alpha or beta secondary structure: its function is unknown. To directly test the deduced topology of the protein, and especially the solubility and stability of its unusual cytoplasmic domain, we have constructed a cDNA expression vector designed to express this domain independently as a soluble protein (designated 5B4cyt) in a heterologous cell system (simian Cos cells). 5B4cyt is indeed soluble, but migrates anomalously on SDS-PAGE under denaturing and reducing conditions as two species of approximately 77 and approximately 80 kDa. In pulse chase experiments, the approximately 77 kDa band chases into the approximately 80 kDa band with a t1/2 of approximately 1 h. The difference in mobility is apparently a consequence of the rapid phosphorylation of the approximately 77 kDa species. The approximately 88 kDa phospho-form is reasonably stable with a t1/2 of approximately 6 h. These results are consistent with the deduced topology of 5B4/NCAM-ld, and demonstrate the feasibility of this experimental approach for exploring the biochemistry and structure of its unusual cytoplasmic domain.