Phosphoglucose isomerase (PGI) was purified from Aspergillus niger and the in vitro kinetic properties of the enzyme were related to its functioning in vivo. A new assay method was developed to study the forward reaction making use of mannitol 1-P dehydrogenase as the coupling enzyme. In this simple assay system mannitol 1-P dehydrogenase converts fructose 6-P and NADH to mannitol 1-P and NAD , respectively. At pH 7.5 the Km for glucose 6-P was 0.48 mM, whereas the Km for fructose 6-P was 0.32 mM. The pentose phosphate pathway intermediates 6-phosphogluconate and erythrose 4-P (E4P) were competitive inhibitors of PGI with Ki values of approximately 0.2 mM and 1 microM respectively. In citric acid producing A. niger mycelium inhibition by 6-phosphogluconate is of minor physiological significance (10% inhibition). Since E4P could not be detected by an existing procedure, a novel assay was developed based on the strong inhibition of PGI by E4P. Although the new assay is very sensitive (detection limit 25 pmol), E4P could still not be detected in metabolite extracts indicating that a very low level of E4P is present in the cells. Using in vitro kinetics and concentrations of intracellular metabolites the in vivo activity of PGI was calculated and closely matched the steady state glycolytic flux observed during citric acid production.