The in vivo dynamics of the pentose phosphate pathway has been studied with transient experiments in continuous culture of Saccaromyces cerevisiae. Rapid sampling was performed with a special sampling device after disturbing the steady state with a pulse of glucose. The time span of observation was 120 s after the pulse. During this short time period the dynamic effect of protein biosynthesis can be neglected. The metabolites of interest (glucose 6-phosphate, NADP, NADPH, 6-phosphogluconate, and MgATP 2& ) we determined with enzymatic assays and HPLC. The experimental observations were then used for the identification of kinetic rate equations and parameters under in vivo conditions. In accordance with results from in vitro studies the in vivo diagnosis supports an ordered Bi-Bi mechanism with noncompetitive inhibition by MgATP 2& for the enzyme glucose-6-phosphate dehydrogenase. In the case of 6-phosphogluconate dehydrogenase an ordered Bi-Ter mechanism with a competitive inhibition by MgATP 2& has been found. Because the MgATP 2& concentration decreases abruptly after the pulse of glucose the inhibitory effect vanishes and the flux through the pentose phosphate pathway increases. This regulation phenomenon guarantees the balance of fluxes through glycolysis and pentose phosphate pathway during the dynamic time period.