Human lymphoid cell lines established by Epstein-Barr viral transformation of peripheral B-lymphocytes from normal subjects and from Fabry patients, were investigated for their ability to biosynthesize neutral glycosphingolipids from [14C]galactose and [14C]glucose as precursors. Galactose was taken up in the presence of high concentrations of glucose and selectively utilised by the cells in the synthesis of galactosphingolipids. The pattern of neutral glycosphingolipids labelled from [14C]galactose was slightly modified with time of labelling in either lymphoid cell line: the first labelled glycosphingolipid was lactosylceramide (LacCer) in the normal line and globotetraosylceramide (GbOse4Cer) in the Fabry line. After labelling for 96 h, a steady state was reached and the percentage of every type of labelled glycosphingolipid was stable in each cell line; however, differences in the neutral sphingolipid composition appeared between the various cell lines. When using radiolabelled glucose as precursor, the major part of the radioactivity was incorporated into neutral lipids and phospholipids; neutral sphingolipids were much less labelled than when using galactose. Catabolism of endogeneous labelled glycosphingolipids (synthesized by the cells during the 'pulse') was studied after cultivating the cells without radiolabelled precursors ('chase'). In the cells from normal subjects, all the neutral glycosphingolipids were slowly degraded (half-life time around 15-25 days for LacCer and GbOse3Cer). In contrast, in a lymphoid line from a Fabry patient, no appreciable degradation of GbOse3Cer occurred during 30 days. This block in the catabolism of GbOse3Cer is in good agreement with the previously reported deficiency of alpha-galactosidase A activity in this Fabry lymphoid cell line [Salvayre, R. et al. (1981) Biochim. Biophys. Acta 659, 445-456] and demonstrates that alpha-galactosidase B does not hydrolyze GbOse3Cer in the living cell (in contrast to the situation in vitro).