Spontaneous Escherichia coli K-12 mutants tolerant to colicin E3 were isolated, and on the basis of their tolerance patterns to 19 kinds of colicins, a new phenotypic class of tolZ mutants was found. The tolZ gene was located between min 77 and 78 on the E. coli K-12 genetic map. The tolZ mutants were tolerant to colicins E2, E3, D, Ia, and Ib, and showed an increased sensitivity to ampicillin, neomycin, and EDTA, but not to deoxycholate; they were able to grow on glucose minimal medium, but not on nonfermentable carbon sources (succinate, acetate, pyruvate, lactate, malate, etc.). The pleiotropic phenotype of the tolZ mutant was due to a single mutation. Both respiration and membrane ATPase activity of the tolZ mutant were normal. The tolZ mutant had a defect in the uptake of proline, glutamine, thiomethyl-beta-D-galactoside, and triphenylmethylphosphonium ion; these uptake systems are driven by an electrochemical proton gradient (delta-mu H+) or a membrane potential (delta psi). In contrast, the uptake of methionine and alpha-methyl-D-glucoside, which is not dependent on delta-mu H+ and delta psi, was normal in the tolZ mutant. Glucose 6-phosphate uptake at pH 5.5, which is driven by a transmembrane pH gradient, in the tolZ mutant was similar to the parent level. These results indicate that the tolZ mutant has a defect in the generation of delta-mu H+ and delta psi.