Cultures derived from a malignant glioma (U-87 MG) were treated with 3 mM dibutyryl cAMP. The treatment resulted in morphological differentiation of the cultures and a decrease in cell proliferation. Biochemically, dibutyryl cAMP treatment caused a general reduction in the concentration of neutral glycosphingolipids in the U-87 MG cells. The concentration of individual neutral glycosphingolipids in the untreated cells was 1.8- to 3.0-fold higher than in cells treated for 72 h with 3 mM dibutyryl cAMP. Cells were labeled with [H-3]galactose to monitor synthesis of the neutral glycosphingolipids. Decreased synthesis was noted in cells treated with dibutyryl cAMP as compared with untreated cells as indicated by decreased uptake of [H-3]galactose label. The ganglioside composition of the cells was essentially unchanged after dibutyryl cAMP treatment.