Four enzymes of the gluconeogenic pathway in Sulfolobus solfataricus were purified and kinetically characterized. The enzymes were reconstituted in vitro to quantify the contribution of temperature instability of the pathway intermediates to carbon loss from the system. The reconstituted system, consisting of phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase and the fructose 1,6-bisphosphate aldolase/phosphatase, maintained a constant consumption rate of 3-phosphoglycerate and production of fructose 6-phosphate over a 1-h period. Cofactors ATP and NADPH were regenerated via pyruvate kinase and glucose dehydrogenase. A mathematical model was constructed on the basis of the kinetics of the purified enzymes and the measured half-life times of the pathway intermediates. The model quantitatively predicted the system fluxes and metabolite concentrations. Relative enzyme concentrations were chosen such that half the carbon in the system was lost due to degradation of the thermolabile intermediates dihydroxyacetone phosphate, glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate, indicating that intermediate instability at high temperature can significantly affect pathway efficiency. Database The mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.mib.ac.uk/ database/kouril/index.html. The investigation and complete experimental data set is available on the SEEK at https://seek.sysmo-db.org/investigations/51. Four enzymes of the gluconeogenic pathway in Sulfolobus solfataricus were purified, kinetically characterized and reconstituted in vitro at 70 °C to quantify the contribution of heat-unstable pathway intermediates to carbon loss from the system. A mathematical model could quantitatively predict the systems fluxes and metabolite concentrations. Approximately half the carbon in the system was lost due to degradation of thermolabile intermediates. © 2013 FEBS.