Introduction of an epoxide function at the C4 position of the glucose moiety of a truncated glucosylceramide (GlcCer) yields two stereoisomeric products namely a gluco- and a galacto-derivative. The effects of both analogues on the [14C]serine incorporation into glycosphingolipids as well as on lactosylceramide (LacCer) synthase activity of primary cultured neurons derived from the telencephalon of 8-day-old chick embryos were investigated. Whereas the galacto-derivative had no effect either on the pattern of labeled glycosphingolipids or on LacCer synthase activity, pretreatment (24 h) of neurons with the gluco-derivative caused a concentration dependent (IC50 = 8 microM) and irreversible decrease of the specific activity of LacCer synthase. As a consequence the biosynthetic glycosphingolipid pattern of cultured neurons treated with the glucoanalogue changed as follows: the radioactive label of all glycosphingolipids biosynthetically derived from GlcCer was decreased while labeling of GlcCer increased significantly. The inhibitory effect of the gluco-derivative on LacCer synthase activity was much less pronounced in vitro. Thus 250 microM analogue caused only about 30% inhibition of enzymatic activity.