The effect of dexamethasone on lipid composition of Epstein-Barr virus transformed human B lymphocytes have been investigated by 31P- and 1H-NMR spectroscopy and compared to the effects due to exogenous sphingosine treatment. Furthermore, the effects of dexamethasone and sphingosine on membrane structure was evaluated by fluorimetry. No significant changes were evidenced in phospholipid composition and in the ratio of unsaturated to total fatty-acid chains. A significant increase in total cholesterol levels was evident at 30 min incubation with dexamethasone or sphingosine; a parallel increase in DPH polarization at 30 min was also demonstrated. TMA-DPH intensity measurements suggest a slowing of vesicular intracellular traffic due to the treatment. The results suggest a dexamethasone- and sphingosine-dependent inhibition of intracellular cholesterol transport.