The effects of neonatal hypothyroidism on the kinetic properties of Na+, K+-ATPase from rat brain microsomes were examined. Neonatal hypothyroidism resulted in decreased Na+, K+-ATPase activity compared to control samples (7.4 ± 1.48 and 29.8 ± 2.30 μmol Pi/h/mg protein, respectively, P < 0.001). Substrate kinetics studies with ATP, Na+ and K+ revealed that there were generalised decreases in Vmax. For ATP, Na+ and K+, activities resolved into two kinetic components in the control group. In hypothyroid animals, the low-affinity component for ATP was absent. The opposite pattern (i.e. an absence of the high-affinity component) was noted for Na+. For K+, although both kinetic components were discernible in neonatal hypothyroid brain microsomes, the Km of the high-affinity component was significantly higher (P < 0.001) compared to control samples. In the control group, the enzyme displayed allosteric behaviour at high concentrations of Mg2+; in hypothyroid animals, the pattern was completely allosteric. The Na+, K+-ATPase enzyme from the hypothyroid brain microsomes bound two molecules of ATP rather than one, unlike in the control animals. Our results thus indicate that neonatal hypothyroidism results in an impairment of microsomal Na+, K+-ATPase activity in the rat brain, together with subtle alterations in the kinetic properties of the enzyme.