Uptake of glycerol was studied in bloodstream and insect forms of the African parasite Trypanosoma brucei using [14C]glycerol in combination with the oil centrifugation technique. Our kinetic measurements revealed that in bloodstream forms glycerol appeared to be transported by two different mechanisms: firstly by a facilitated-diffusion carrier showing a Km of 0.17 mM and a Vmax of 44 nmol 10(-8) cells min(-1) that predominates at low glycerol concentrations, and secondly by simple diffusion. The effects induced by various inhibitors suggest that uptake is neither sodium dependent nor proton-motive-force driven. The saturable component of transport was phloretin and cytochalasin B sensitive and could also be inhibited by the substrate analogue glyceraldehyde, which led to a 74% decrease in glycerol uptake. In insect forms, however, glycerol is taken up by simple diffusion only. Uptake was insensitive to mercury ions and was not influenced by a variety of different channel inhibitors. Our data show that in T brucei glycerol transport across the plasma membrane occurs by simple diffusion. In addition, bloodstream forms express a carrier protein which promotes a rapid transport at low glycerol concentrations. Expression of this transport protein may account for a selective secretion of intracellular glycerol which otherwise could become toxic for the parasite due to its specific compartmentation of glycolysis.