Fructose was shown to be phosphorylated by a specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) in Xanthomonas campestris pv. campestris. Transposon mutagenesis of X. campestris was performed and two mutants affected in growth on fructose were isolated. Both mutants were deficient in PTS activity. Comparison of the rate of uptake and phosphorylation of fructose in the wild-type and in the mutant strains revealed the presence of a second fructose permeation and phosphorylation pathway in this bacterium: an unidentified permease coupled to an ATP-dependent fructokinase. One of the two mutants was also deficient in fructokinase activity. Chromosomal DNA fragments containing the regions flanking the transposon insertion site were cloned from both mutant strains. Their physical study revealed that the insertion sites were separated by 1.4 kb, allowing the reconstruction of a wild-type DNA fragment which complemented one of the two mutants. The region flanking the transposon insertion site was sequenced in one of the mutants, showing that the transposon had interrupted the gene encoding the fructose EII. The mutant strains also failed to utilize mannose, sucrose and mannitol, suggesting the existence of a branch point between the metabolism of fructose and of these latter carbohydrates.