In previous studies examining the role of glucocorticoids and thyroid hormone on the maturation of the Na⁺/H⁺ antiporter (NHE3), we found attenuation in the maturational increase in proximal tubule apical Na⁺/H⁺ antiporter activity, but no change in NHE3 mRNA abundance in either glucocorticoid deficient or hypothyroid rats. In addition, prevention of the maturational increase in either hormone failed to totally prevent the maturational increase in Na⁺/H⁺ antiporter activity. We hypothesized that one hormone played a compensatory role when other was deficient. The present study examined whether combined deficiency of thyroid and glucocorticoid hormones would completely prevent the maturation of Na⁺/H⁺ antiporter. Adrenalectomy was performed in nine-day-old hypothyroid Sprague-Dawley rats, a time before the normal postnatal maturational increase in these hormones. Nine-day and 30-day-old adrenalectomized (ADX)-hypothyroid rats had comparable NHE3 mRNA abundance, which was 5-10 fold less than 30-day old ADX-hypothyroid rats that received corticosterone-thyroxine replacement and 30-day-old sham control rats (P<0.05). Brush border membrane NHE3 protein abundance was comparable in 9-day-old and 30-day ADX-hypothyroid groups and approximately 20-fold lower than both the 30-day replacement and 30-day sham groups (P<0.05). Similarly, the replacement and sham groups had higher sodium-dependent proton secretion than 9-day-old, and 30-Day-ADX-hypothyroid groups (P<0.05). We conclude that combined deficiency of both hormones totally prevents the maturational increase of NHE3 mRNA and protein abundance and Na⁺/H⁺ antiporter activity.