We used 31P-NMR to study the action of the drug ouabain, a digitalis glycoside, on the ecto-nucleotidase catalysed reaction pathway: [formula: see text] Even under conditions where it was impossible to resolve all resonances from the individual species, the rate constants for the individual steps may be evaluated by a chi 2 fit of equations describing the global kinetics to the NMR data. The fitted values of the reaction rate constants show no inhibition of any step in the reaction pathway, confirming previous results. This improved kinetic method can be used to evaluate the effect of different substrates, ions and drugs, both in intact or pathologically altered organs and in tissue subcellular fractions.