A method for the quantification of intracellular metabolic flux distributions from steady-state mass balance constraints and from the constraints posed by the measured 13 C labeling state of biomass components is presented. Two-dimensional NMR spectroscopy is used to analyze the labeling state of cell protein hydrolysate and cell wall components. No separation of the biomass hydrolysate is required to measure the degree of 13 C 13 C coupling and the fractional 13 C enrichment in various carbon atom positions. A mixture of [1-13 C]glucose and uniformly labeled [ 13 C 6 ]glucose is applied to make fractional 13 C enrichment data and measurements of the degree of 13 C 13 C coupling informative with respect to the intracellular flux distribution. Simulation models that calculate the complete isotopomer distribution in biomass components on the basis of isotopomer mapping matrices are used for the estimation of intracellular fluxes by least-squares minimization. The statistical quality of the estimated intracellular flux distributions is assessed by Monte Carlo methods. Principal component analysis is performed on the outcome of the Monte Carlo procedure to identify groups of fluxes that contribute major parts to the total variance in the multiple flux estimations. The methods described are applied to a steady-state culture of a glucoamylase-producing recombinant Aspergillus niger strain.