The influence of dietary fatty acids on Na+ sensitivity and ouabain affinity of Na+/K+-ATPase isoenzymes of whole brain membranes were studied in weaned rats fed for two generations with diets either devoid of alpha-linolenic acid (sunflower oil diet) or rich in alpha-linolenic acid (soya oil diet). The (n - 3) deficiency induced by the sunflower oil diet led to an increase in the (n - 6)/(n - 3) molar ratio in whole brain membranes. Na+/K+-ATPase isoenzymes were discriminated on the basis of their differential affinities for ouabain. In rats fed sunflower oil diet, the ouabain titration displayed three inhibitory processes with markedly different affinities: low affinity (alpha1); high affinity (alpha2); and very high affinity (alpha3). Membranes of rats fed soya oil diet exhibited only two inhibitory processes, i.e., low affinity (likely alpha1 + alpha2) and high affinity (likely alpha2 + alpha3) with the low affinity form intermediate between the sunflower alpha1 and alpha2 forms, and the high affinity form intermediate between the sunflower alpha2 and alpha3 forms. In fact, the Na+ response shows that the three isoenzymes have different Na+ sensitivities. Regardless of the diet, alpha1 has a similar Na+ sensitivity (less than 1 mM), whilst alpha2 and alpha3 are more sensitive in soya oil membranes compared to sunflower oil membranes (5.1 vs. 7.2 mM and about 11 vs. 22.5 mM, respectively). Thus, sodium appears to be a better criterion of heterogeneity than ouabain.