We have cloned the pps gene, coding for PEP synthase, of Escherichia coli. PEP synthase catalyses the ATP-dependent conversion of pyruvate into phosphoenol-pyruvate and is required for gluconeogenesis. The pps gene was cloned by an in vivo cloning method using a mini-Mulac bacteriophage containing a plasmid replicon. Upon expression of the cloned pps gene in the maxicell system a protein with an apparent molecular weight of 84 kDa was synthesized. The position of the pps gene of the plasmid was localized by restriction analysis of isolated transposon insertions and the determination of the PEP synthase activities of the different clones. An operon fusion between the pps gene and the galK gene was constructed. Measurements of the galactokinase activity in Salmonella typhimurium galK and galK fruR mutants showed that the transcription of the pps gene requires the presence of FruR, the repressor of the PEP: fructose phosphotransferase system (PTS) in E. coli and S. typhimurium. To test whether the components of the Fructose PTS, in particular FPr, are involved in the expression of the pps gene, we investigated a S. typhimurium galK strain, containing the fusion plasmid, in which the chromosomal fru operon was inactivated by a transposon insertion. Measurements of the galactokinase activity showed that the absence of the Fructose PTS proteins has no significant influence on the regulation of the pps gene.