There are three main routes for the utilization of fructose by Escherichia coli. One (Route A) predominates in the growth of wild-type strains. It involves the functioning of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and a fructose operon, mapping at min. 48.7, containing genes for a membrane-spanning protein (fruA), a 1-phosphofructose kinase (fruK) and a diphosphoryl transfer protein (fruB), under negative regulation by a fruR gene mapping at min. 1.9. A second route (Route B) also involves the PTS and membrane-spanning proteins that recognize a variety of sugars possessing the 3,4,5-D-arabino-hexoseconfiguration but with primary specificity for mannose(manXYZ), mannitol (mtlA) and glucitol (gutA) and which, if over-produced, can transport also fructose. A third route (Route C), functioning in mutants devoid of Routes A and B, does not involve the PTS: fructose diffuses into the cell via an isoform (PtsG-F) of the major glucose permease of the PTS and is then phosphorylated by ATP and a manno(fructo)kinase (Mak+) specified by a normally cryptic 1032 bp ORF (yajF) of hitherto unknown function (Mak-o), mapping at min. 8.8 and corresponding to a peptide of 344 amino acids. Conversion of the Mak-o to the Mak+ phenotypeinvolves an A24D mutation in a putative regulatory region.