The Escherichia coli otsBA operon, located at min 42, was sequenced and shown to encode a 29.1-kDa trehalose-6-phosphate phosphatase (OtsB) and a 53.6-kDa trehalose-6-phosphate synthase (OtsA). Both proteins display sequence homology with subunits of the Saccharomyces cerevisiae trehalose-6-phosphate synthase/phosphatase complex, which is made up of the subunits TPS1, TPS2 and TPS3 (TSL1). OtsA has homology to the full-length TPS1, the N-terminal part of TPS2 and an internal region of TPS3 (TSL1). OtsB has homology to the C-terminal part of TPS2, but no homology to the other subunits. Primer extension analysis showed only one transcription start point upstream from otsB and one upstream from otsA, regardless of the growth conditions tested. The start codons of the otsB and otsA genes were established by N-terminal sequence determination of the proteins. The 3' end of the otsB coding region overlaps the 5' end of the otsA coding region by 23 nucleotides. The araH gene is located directly upstream from otsBA, and otsB may be identical to pexA.