Using dodecyl N-acetylglucosaminide (GlcNAc-C12) as a saccharide primer, we investigated the biosynthetic changes of neolacto-series glycosphingolipids (GSLs) in mouse embryonal carcinoma F9 cells during differentiation induced by retinoic acid plus dibutyryl cyclic AMP (RA/dbcAMP). In the differentiated cells, the glycosylation of GlcNAc-C12 was greatly enhanced. The sugar compositions of glycosylated primers were assigned as Hex-GlcNAc, [Hex](2)-GlcNAc, [Hex](2)[HexNAc]-GlcNAc, and [NeuAc][Hex]-GlcNAc by liquid chromatography-tandem mass spectrometry. The detection of augmented biosynthesis of endogenous sialylparagloboside indicated that [NeuAc][Hex]-GlcNAc was predicted to be the non-reducing end trisaccharide of sialylparagloboside. The transcription of B3gnt5, B4galt1, Ggta1, Fut4 and St3gal6, encoding glycosyltransferases involved in the neolacto-series glycosphingolipids biosynthesis, was increased, whereas that of Fut9 and St6galI was decreased after RA/dbcAMP treatment. Furthermore, the sialyltransferase activity of ST3GalVI sialylating paragloboside was enhanced with the increase in St3gal6 expression. Since most stage-specific embryonic antigen-1 (SSEA-1) active determinants are carried by glycoproteins in F9 cells, the changes in glycolipid metabolism do not seem to be closely related to loss of cell surface SSEA-1 expression upon F9 differentiation. These results indicate that RA/dbcAMP treatment activates the biosynthesis of neolacto-series GSL and enhances sialylation of paragloboside in F9 cells with down-regulation of Fut9 expression.