This contribution addresses the identification of metabolic fluxes and metabolite concentrations in mammalian cells from transient C-13-labeling experiments. Whilst part I describes experimental set-up and acquisition of required metabolite and C-13-labeling data, part II focuses on setting up network models and the estimation of intracellular fluxes. Metabolic fluxes were determined in glycolysis, pentose-phosphate pathway (PPP), and citric acid cycle (TCA) in a hepatoma cell line grown in aerobic batch cultures. In glycolytic and PPP metabolite pools isotopic stationarity was observed within 30 min, whereas in the TCA cycle the labeling redistribution did not reach isotopic steady state even within 180 min. In silico labeling dynamics were in accordance with in vivo C-13-labeling data. Split ratio between glycolysis and PPP was 57%:43%; intracellular glucose concentration was estimated at 101.6 nmol per 10(6) cells. In contrast to isotopic stationary C-13-flux analysis, transient C-13-flux analysis can also be applied to industrially relevant mammalian cell fed-batch and batch cultures.