Cells of Saccharomyces cerevisiae are able to transport trehalose against a concentration gradient, without efflux or counterflow of the labeled substrate. Uptake was inhibited by uncouplers, acetic acid, and organic mercury compounds. The addition of trehalose resulted in alkalinization of the medium. The ratio of H+ depletion to trehalose uptake by yeast cells was approximately 1:1, which indicates the existence of a trehalose-H+ symporter in these cells. The optimum pH for this active H+-trehalose symport was 5.0, and both the Km and the Vmax were negatively affected by increasing or decreasing the extracellular pH from its optimum value. Kinetic studies showed the existence of at least two different trehalose transport activities in yeast cells: a high-affinity H+-trehalose symporter (Km = 4 mM), and a low-affinity transport activity (Km > 100 mM) that could be a facilitated diffusion process. The high-affinity H+-trehalose symporter was repressed by glucose, whereas the low-affinity uptake was constitutively expressed in S. cerevisiae.