Escherichia coli can use the nonreducing disaccharide trehalose as a sole source of carbon and energy. Trehalose transport into the cell is mediated via the phosphotransferase system, and a mutant depleted in the nonspecific proteins enzyme I, HPr, and enzyme IIIGlc of this system was not only unable to grow on glucose or mannitol but also was strongly reduced in its ability to grow on trehalose. A pseudorevertant (PPA69) of such a deletion mutant was isolated that could again grow on glucose but not on mannitol. This revertant could now also use trehalose as a carbon source due to a constitutive galactose permease. PPA69 was subjected to Tn10 insertional mutagenesis, and a mutant (UE5) was isolated that no longer could use trehalose as a carbon source but could still grow on glucose. UE5 lacked a periplasmic trehalase that was present in PPA69. P1-mediated transduction of this Tn10 insertion (treA::Tn10) into a pts+ wild-type strain (MC4100) had no effect on the ability of MC4100 to grow on trehalose but resulted in loss of the periplasmic trehalase activity. The Tn10 insertion was mapped at 26 min on the E. coli linkage map and was 3% cotransducible with trp, in the order treA::Tn10, trp, cys. Trehalase activity in MC4100 was not induced by growth in the presence of trehalose but increased by about 10-fold when 0.6 M sucrose was added to minimal growth medium. Using the in vivo mini-Mu cloning system and growth on trehalose as selection, we cloned the treA gene. A 9-kilobase EcoRI fragment containing treA was subcloned into pBR322. Strains carrying this plasmid (pTRE5) contained about 100-fold higher periplasmic trehalase activity than PPA69 or MC4100. Using polyacrylamide gel electrophoresis, we found a protein of molecular weight 58,000 among the periplasmic proteins of the pTRE5-carrying strain that was absent in UE5. This protein was purified by ammonium sulfate precipitation and DEAE-Sepharose ion-exchange chromatography and contained all the trehalase activity. Minicells containing the treA+ plasmid produced, in addition to three other proteins, the 58,000-dalton protein. Thus, the plasmid carries the structural gene for the periplasmic trehalase and not just a gene involved in the regulation of the enzyme.