N-Acylaminoethyl lactosides as lactosylceramide analogs as well as n-alkyl lactosides were examined for their ability to prime glycosphingolipid (GSL) synthesis in mouse melanoma B16 cells. Using compounds radiolabeled in a galactose residue and having nondegradable thioglucosidic linkages in lactoside, direct glycosylation was shown to occur at the terminal galactose residue of lactosides subsequent to uptake by cells and dissemination into Golgi compartments. B16 cells took in lactosides temperature-dependently to the point of saturation. All lactosides were taken up and glycosylated by B16 cells. C8-lactosides could not settle on the plasma membrane, while C16-lactosides remained within the cells. Glycosylation in all cases was cellular GSL-specific, suggesting the involvement of glycosyltransferases in GSL synthesis during glycosylation of lactosides. The priming of GSL synthesis by lactosides inhibited the cell surface expression of endogenous GM3 in B16 cells. Lactosylceramide analogs are thus shown useful as primers for glycosylation and to modify GSL expression, and these features should facilitate clarification of the functions of GSLs which have yet to be elucidated.