In Salmonella enterica serovar Typhimurium, the genomic island GEI4417/4436 has recently been identified to be responsible for myo-inositol (MI) utilization. Here, two of the four island-encoded permeases are identified as the MI transporters of this pathogen. In-frame deletion of iolT1 (STM4418) led to a severe growth defect, and deletion of iolT1 (STM4419) to a slight growth defect in the presence of MI. These phenotypes could be complemented by providing the putative transporter genes in trans. Bioluminescence-based reporter assays demonstrated a strong induction of their promoters P(iolT1) and P(iolT2) in the presence of MI but not of glucose. Deletion of iolR, which encodes the negative regulator of most genes involved in MI degradation, resulted in upregulation of P(iolT1) and P(iolT2), indicating that the expression of IolT1 and IolT2 is repressed by IolR. This finding was supported by bandshift assays using purified IolR. Both transporters are located in the membrane when expressed in Escherichia coli. Heterologously expressed IolT1 had its optimal activity at pH 5.5. Together with the strongly reduced MI uptake in the presence of protonophores, this indicates that IolT1 operates as a proton symporter. Using myo-[1,2-[(3)H](N)]inositol, a saturable uptake activity of IolT1 with a K(m) value between 0.49 and 0.79 mM was determined in DH5alpha expressing IolT1, in S. enterica serovar Typhimurium strain 14028, and in mutant 14028 DeltaiolT2. Phylogenetic analysis of IolT1 identified putative MI transporters in Gram-negative bacteria also able to utilize MI.