In the developing rat cerebrum, the level of different isoforms of Na-K-ATPase mRNA increases significantly during the first three postnatal weeks, which represent the critical period of synaptogenesis and myelination-the two thyroid hormone-sensitive maturational events. To determine the possible functional relationship of these isoforms with maturational events in the developing brain and their mode of regulation by T3, we have examined the effect of hypothyroidism on the expression of the different alpha-isoforms (alpha 1, alpha 2, and alpha 3) of Na-K-ATPase mRNA covering the first 3 wk of postnatal development. Quantitation of these mRNAs from cerebra of 1-, 5-, 10-, 15-, and 20-d-old normal and hypothyroid rats by Northern blot analysis indicate that alpha 3 mRNA is not only predominantly expressed throughout this entire period of study but also represents the species which is most severely affected in the hypothyroid brain. The relative sensitivity for the expression of these mRNAs to T3 were alpha 3 > alpha 1 > alpha 2. These results, together with the report of predominant expression of the alpha 3 isoform in neuronal cells, suggest specific functional involvement of this isoform with the decisive maturational events in the rat brain. Kinetic studies on in vivo induction of Na-K-ATPase alpha-mRNAs by T3 in the 15-d-old hypothyroid rat shows clear stimulation of all the isoforms within 1 h of the administration of the optimal dose (200 micrograms T3/100 g body wt) suggesting a direct, possibly transcriptional effect of the hormone on the expression of these genes.