Aldosterone and thyroid hormone regulation of Na,K-pump biosynthesis has been examined in the distal colon epithelium of rabbits. Qualitative analysis of α-subunit isoform distribution (α1, α2, α3) detected only the α1-mRNA in the distal colon epithelium and outer renal medulla, while all three isoforms were observed in rabbit brain. A low-sodium diet led to a rise in serum aldosterone from 0.6 n m to 1.4-1.9 n m and an increase of α1-mRNA to 162%, β1-mRNA to 120%, and the number of Na,K-pump units as determined by specific [³H]-ouabain binding to 182% of control by the second day of the diet. While aldosterone levels remained elevated, a spontaneous decrease in serum levels of T₃ and T₄ to 50-60% of control from the third day of the diet was followed by downregulation of β1-mRNA to 55-67%, α1-mRNA to 63-105%, and of [³H]-ouabain binding to 103% of control, suggesting that a reduced rate of synthesis of the β1-subunit is rate limiting for Na,K-pump biosynthesis. Substitution with T₃ (10 μg/kg) at the seventh day with transient restoration of serum T₃ to control levels, led to rapid accumulation of β1-mRNA to 152%, of α1-mRNA to 135%, and of the number of Na,K-pump units to 153% of control. This is consistent with thyroid hormone having a permissive role for the aldosterone stimulation of Na,K-pump biosynthesis. Reduced rates of β-subunit transcription due to low thyroid hormone levels appear to provide a mechanism for escape from the effect of hyperaldosteronemia on the number of Na,K-pump units.