Myelin basic protein (MBP) from shark (Chondricthyes) consists of a simpler mixture of charge isomers than human MBP. About two‐thirds of the total amount applied to a CM‐52 cellulose cation‐exchange column was recovered in the unbound fraction of the column; the remaining one‐third bound to column and was eluted as a single OD₂₈₀ peak. This bound material did not show the usual pattern of charge microheterogeneity found with human or bovine MBP. The unbound fraction was composed of a high molecular weight protein (55–60 kDa), which constituted most of this protein fraction and a low molecular weight protein (∼ 18 kDa). The amino acid composition of our unbound fraction was similar to that reported earlier. The Glx (glutamic acid + glutamine) was increased about threefold whereas the Arg content was only about 25% of that of the 18.5 kDa variant of bovine or human origin. The presence of hydroxyproline (1.2 residues/100) in this protein was noteworthy, identification of which was achieved by amino acid analysis in two different systems and by mass spectrometry. In the precolumn derivatization method, hydroxyproline eluted at 2.7 min; in the postcolumn derivatization method it eluted at 12.2 min. Identification of hydroxyproline was completed by fast atom bombardment‐mass spectral analysis. The effect of hydroxyproline on the secondary structure of this protein is being studied. Verification that this high molecular weight protein contained MBP sequences within its primary structure was confirmed by immunological methods. Good reactivity, comparable to that found with human MBP was observed in the enzyme‐linked immunosorbent assay with an antibody (S‐53) raised in rats against the synthetic peptide GSLPQKAQRPQDEN. The fraction that bound to the CM‐52 cellulose column and was eluted with a salt gradient corresponded in net charge to component 5 of the bovine or human MBPs as deduced by alkaline gels.