We previously demonstrated that there are developmental changes in proximal tubule Na(+)/H(+) exchanger (NHE) activity. There is a maturational increase in postnatal brush-border membrane (BBM) vesicle NHE3 protein abundance and decrease in NHE8 protein abundance. The purpose of this study was to determine whether thyroid hormone plays a role in the rat renal maturational isoform switch from NHE8 to NHE3 and whether thyroid hormone regulates NHE8. Administration of thyroid hormone to neonatal rats, before the normal postnatal increase in serum thyroid hormone levels at 3 wk of age, resulted in a premature increase in NHE3/beta-actin BBM protein abundance and mRNA abundance. Thyroid hormone also caused a premature decrease in BBM NHE8/beta-actin protein abundance, whereas there was no change in mRNA expression (standardized to 28s). Rats made hypothyroid from birth were studied at 28 days, after the normal maturational increase in thyroid hormone. In these hypothyroid adult rats, the maturational increase in BBM NHE3 protein abundance and NHE3 mRNA expression was prevented. In contrast, the developmental decrease in BBM NHE8 protein abundance was prevented in hypothyroid adults, but mRNA expression was unchanged in hypothyroid rats. To determine whether the effect of thyroid hormone was due to a direct epithelial effect, we studied normal rat kidney cells in culture. We recently showed that this cell line expresses NHE8, but does not express NHE3. Thyroid hormone caused a decrease in surface expression of NHE8, determined by biotinylation, but total cellular abundance remained unchanged. NHE8 activity, measured as the sodium-dependent rate of intracellular pH recovery from an acid load, was less with thyroid treatment than control. In conclusion, thyroid hormone plays a potential role in the developmental isoform change from NHE8 to NHE3 and decreases NHE8 activity.