The protein components of the white spot syndrome virus (WSSV) virion have been well established by proteomic methods, and at least 39 structural proteins are currently known. However, several details of the virus structure and assembly remain controversial, including the role of one of the major structural proteins, VP26. In this study, Triton X-100 was used in combination with various concentrations of NaCl to separate intact WSSV virions into distinct fractions such that each fraction contained envelope and tegument proteins, tegument and nucleocapsid proteins, or nucleocapsid proteins only. From the protein profiles and Western blotting results, VP26, VP36A, VP39A, and VP95 were all identified as tegument proteins distinct from the envelope proteins (VP19, VP28, VP31, VP36B, VP38A, VP51B, VP53A) and nucleocapsid proteins (VP664, VP51C, VP60B, VP15). We also found that VP15 dissociated from the nucleocapsid at high salt concentrations, even though DNA was still present. These results were confirmed by CsCl isopycnic centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry, by a trypsin sensitivity assay, and by an immunogold assay. Finally, we propose an assembly process for the WSSV virion.