The stage of lactation is one of the most important factors that influence milk composition. Changes in fatty acids from triacylglycerols and phospholipids have already been reported. In this study, we looked for a lactational change in the ganglioside lipid moiety since ganglioside contents and patterns vary strongly with stage of lactation. Individual gangliosides from four stages were isolated, methanolized to cleave the bonds between individual constituents, and derivatized for gas-liquid chromatography and gas chromatography/mass spectrometry analyses. Ceramide components, both fatty acids (as methyl esters derivatives) and long-chain bases, were identified and quantified. The results pointed to a marked change in ceramide from colostrum to milk that was characterized by a dramatic decrease in saturated and the longest-chain fatty acids as well as an increase in 18:1 and 18:2. The major long-chain base along lactation was a recently described structure, 3-ethoxy-15:0 sphinganine. Other new long-chain base structures appeared in these gangliosides. All these changes suggest differences in the fluidity of the fat globule membrane, reflecting physiological variations in cows with respect to milk production.