Eadie-Hofstee plots of glycerol uptake in wild-type Saccharomyces cerevisiae W303-1A grown on glucose showed the presence of both saturable transport and simple diffusion, whereas an fps1delta mutant displayed only simple diffusion. Transformation of the fps1delta mutant with the glpF gene, which encodes glycerol transport in Escherichia coli, restored biphasic transport kinetics. Yeast extract-peptone-dextrose-grown wild-type cells had a higher passive diffusion constant than the fps1delta mutant, and ethanol enhanced the rate of proton diffusion to a greater extent in the wild type than in the fps1delta mutant. In addition, the lipid fraction of the fps1delta mutant contained a lower percentage of phospholipids and a higher percentage of glycolipids than that of the wild type. Fps1p, therefore, may be involved in the regulation of lipid metabolism in S. cerevisiae, affecting membrane permeability in addition to fulfilling its specific role in glycerol transport. Simultaneous uptake of glycerol and protons occurred in both glycerol- and ethanol-grown wild-type and fps1delta cells and resulted in the accumulation of glycerol at an inside-to-outside ratio of 12:1 to 15:1. Carbonyl cyanide m-chlorophenylhydrazone prevented glycerol accumulation in both strains and abolished transport in the fps1delta mutant grown on ethanol. Likewise, 2,4-dinitrophenol inhibited transport in glycerol-grown wild-type cells. These results indicate the presence of an Fps1p-dependent facilitated diffusion system in glucose-grown cells and an Fps1p-independent proton symport system in derepressed cells.