The ganglioside and neutral glycosphingolipid composition of fetal and adult sheep colonic mucosa were characterized and compared. Mono- and tetrahexosylceramide were the major neutral glycolipids of both fetal and adult colons. Adult, but not fetal, mucosa also possessed di- and trihexosylceramide. Similarly, GD1a, GM3 and GM2 were found to be the principal gangliosides in fetal and adult tissue. Adult colonic mucosa possessed significant amounts of GT1a not present in fetal tissue. Analysis of the hydroxy and nonhydroxy fatty acids as well as of the long chain bases of the major glycosphingolipids revealed differences between these lipophilic components of glycolipids in fetal and adult colonic mucosa. The present results, therefore, indicate that both quantitative and qualitative differences in glycosphingolipid composition exist between fetal and adult sheep colonic mucosa.