We report the isolation and initial characterization of mutations in the newly described eutF locus of Salmonella typhimurium LT2. Mutations in eutF render a strain unable to utilize ethanolamine as a source of carbon and/or energy and impair growth on ethanolamine as a sole nitrogen source. Strains carrying eutF mutations exhibit a 2-order-of-magnitude decrease in transcription of the unlinked eutDEABCR operon (50 min), which codes for the enzymes needed to catabolize ethanolamine; have only 10% of the ethanolamine ammonia-lyase activity found in the wild type; and show a marked reduction in the rate of ethanolamine uptake. Deletion mapping and three-factor cross analysis results are consistent with the gene order cobA trp eutF tonB at 34 min on the linkage map. We discuss two possible roles for the EutF protein: (i) as an ethanolamine permease or (ii) as a transcription factor required for the expression of the eutDEABCR operon.