We describe the molecular cloning of the gal operon of Salmonella typhimurium LT2 and the localization of the gal promoter and the genes galE, galT, and galK. The order of the genes and the direction of transcription was the same as in Escherichia coli K12. A restriction enzyme map of the operon was obtained, and the approximate termini of the gal genes were located by using transposon insertion mutagenesis and minicell analysis. We constructed a plasmid that contained a defined 0.4-kilobase deletion in the galE but still expressed galT and galK activities from the gal promoter. This galE deletion was recombined into the chromosomal gal operons of S. typhimurium and Salmonella typhi Ty2. The resulting strains were vigorous, nonreverting galE mutants that were sensitive to galactose-induced lysis at 0.2 mM galactose. The S. typhimurium galE derivatives were avirulent and protective in mice.