The estimation of intracellular fluxes of mammalian cells using only mass balances of the relevant metabolites is not possible because the set of linear equations defined by these mass balances is underdetermined. in order to quantify fluxes in cyclic pathways the mass balance equations can be complemented with several constraints : (1) the mass balances of co-metabolites, such as ATP or NAD(P)H, (2) linear objective functions, (3) flux data obtained by isotopic-tracer experiments. Here, these three methods are compared for the analysis of fluxes in the primary metabolism of continuously cultured hybridoma cells. The significance of different theoretical constraints and different objective functions is discussed after comparing their resulting flux distributions to the fluxes determined using (CO2)-C-13 and C-13-lactate measurements of 1-C-13-glucose-fed hybridoma cells. Metabolic fluxes estimated using the objective functions "maximize ATP" and "maximize NADH" are relatively similar to the experimentally determined fluxes. This is consistent with the observation that cancer cells, such as hybridomas are metabolically hyperactive, and produce ATP and NADH regardless of the need for these cofactors.