Cerebrosides, mainly glucocerebrosides and galactocerebrosides, are the simplest glycosphingolipids (GLSs) and known to be the biosynthetic precursors of most other GLSs. 1) While glucocerebrosides have been isolated from various starfishes, occurrence of galactocerebrosides seems to be limited to three species: Stellaster equestris, 2) Culcita novaeguineae, 3) and Oreaster reticulatus. 4) In our continuing research on GLSs from echinoderms, a galactocerebroside molecular species (PNC-1) was purified from the starfish Protoreaster nodosus (kobuhitode in Japanese) collected in Okinawa. Reversed-phase HPLC separation of PNC-1 afforded sixteen new galactocerebrosides (3-7, 9-17, 19, 21), along with five known galactocerebrosides (1, 2, 8, 18, 20). Further, we developed one-pot GC-MS analysis for cerebrosides which consist of phytosphingosinetype long chain base (LCB). Generally, the trimethylsilyl (TMS) derivatives of LCB are used for GC-MS analysis and give a useful structural information, 5) however, the preparation of phytosphingosine-type LCB from the parent cerebroside is in a quite low yield and not quantitative, because the glycosidic linkage between sugar and LCB is hard to cleavage against methanolysis. One-pot reaction following mathanolysis and periodate oxidation, and subsequent GC-MS analysis gave efficient structural information. In this paper, the isolation and structure elucidation of galactocerebroside molecular species (PNC-1) and constituent cerebrosides (1-21) of PNC-1 from the starfish Protoreaster nodosus are described.Isolation and Structure of PNC-1 The lipid fraction, obtained from CHCl 3 /MeOH extract of pyloric caeca, which was dissected from the fresh material of the starfish P. nodosus, was subjected to repeated silica gel, reversed-phase and Sephadex LH-20 column chromatography to give a less polar GLS, named PNC-1, showing a single spot on silica gel TLC plate.The positive ion FAB-MS spectrum of PNC-1 exhibited a series of homologous pseudomolecular ion peaks [MϩNa] )] in the total correlation spectroscopy (TOCSY) spectrum. Thus, a structural fragment from H 2 -1 to H 2 -6 was established, suggesting the LCB of ceramide was phytosphingosine-type. Analysis of COSY and TOCSY spectra revealed another structural fragment (H-2Ј to H 2 -4Ј) in the fatty acid part of ceramide. H-2Ј (d H 4.52) signal showed a heteronuclear multiple-bond correlation spectroscopy (HMBC) cross peak with the amide carbon signal [d C 175.7 (C-1Ј)], indicating the presence of a-hydroxy fatty acid. Further, H-2 (d H 5.18) signal in LCB showed a cross peak with amide carbon (C-1Ј), and the ceramide moiety was depicted as shown in Fig. 1.Identification of the compositions of fatty acid and LCB of ceremide moiety was conducted by GC-MS analysis following methanolysis and periodate oxidation of PNC-1 (see Experimental). The mixture of fatty acid methyl esters (FAMEs) obtained from methanolysis and a mixture of long chain aldehydes generated by periodate oxidation were simultaneously subjected to GC-MS, which revealed ...