The co-transport of uracil and protons through the plasma membrane of the yeast Saccharomyces cerevisiae is mediated by a specific permease encoded by the FUR4 gene. The uracil permease is a multi-spanning membrane protein that follows the secretory pathway to the plasma membrane. Recent experimental data led to the proposal of a two-dimensional model of its topology. A spontaneous mutant corresponding to the substitution of Lys-272 by glutamic acid was obtained. The influence of this mutation was studied by comparing the wild-type and mutant permeases produced in a strain carrying a chromosomal deletion of the FUR4 gene. The mutant permease is correctly targeted to the plasma membrane and its stability is similar to that of the wild-type permease. The uptake parameters for the mutant permease were impaired and showed an approximately 65-fold increase of apparent K(m) and a decrease in apparent Vmax. Equilibrium binding measurements with enriched plasma membrane preparations showed an approximately 70-fold increase in apparent Kd in the mutant, whereas its Bmax. was similar to that of the wild type. Lys-272 is fully conserved in the uracil permease family and is predicted to lie in the fourth transmembrane segment of the protein. It seems to be essential for both efficient uracil binding and translocation.