A new approach is proposed for investigating the mechanism of metabolite synthesis in cells. This method, based on the competition between various substrates, allows the flux along a pathway, which is normally independent of the concentration of the corresponding precursor in the external medium, to be divided into partial fluxes. In particular, the mechanism deoxy-trehalose synthesis in glucose-grown repressed Saccharomyces cerevisiae was studied, by 1H-NMR spectroscopy, using the competition between 2-deoxy-D-glucose (dGlc) and 2-fluoro-deoxy-D-glucose (FdGlc) with respect to hexokinase. S. cerevisiae cells, suspended in a standard pyrophosphate medium containing about 5 x 10(7) cells/ml, were incubated with 30 mM glucose and various concentrations of dGlc and FdGlc. Apart from dGlc6P and FdGlc6P, trehalose and the dissacharides relative to dGlc, i.e. dideoxy-trehalose (dGlc-dGlc) and deoxytrehalose (dGlc-Glc), are observed while their analogues relative to FdGlc (FdGlc-FdGlc, FdGlc-Glc) are surprisingly absent. For the same external concentration of dGlc and FdGlc, the internal concentration of FdGlc6P is about three times larger than that of dGlc6P. The ratio of the FdGlc6P and dGlc6P concentrations is independent of the incubation times and proportional to the FdGlc and dGlc concentrations in the suspension. The dGlc6P concentration can thus be reduced at will by increasing the [FdGlc]/[dGlc] ratio. Under these conditions, the dGlc-Glc concentration was found to vary linearly with that of dGlc6P. The present data clearly show that deoxy-trehalose is not synthesized from UDP-dGlc and Glc6P but from UDP-Glc and dGlc6P. This conclusion was also confirmed by an experiment in which S. cerevisiae cells were previously charged with dGlc6P and then incubated with glucose.