Repeated administration of thyroxine (T4) in vivo (2 microgram/100 g body wt for 6 days) lowered by 2.3 times (P less than 0.025, df = 18) the basal rate of Na+ transport measured by the short-circuit current (SCC) in vitro in the urinary bladder of the toad (Bufo marinus). This difference was not accounted for by a change in the plasma aldosterone concentration. Moreover the response of the SCC to aldosterone in vitro was markedly inhibited in bladders from T4-treated animals (P less than 0.001, df = 18). These findings raised the possibility of a direct interaction between thyroid hormone and aldosterone in the target cell. The effects of L-triiodothyronine (T3) and aldosterone were examined in vitro. T3 alone (60 nM) had no significant effect on the base-line SCC (deltamuA = -14 +/- 11 (SE) muA per hemibladder; P greater than 0.3, n = 10). By contrast, T3 (60 nM) inhibited the response of the SCC to aldosterone from 6 to 8 h after its addition (deltamuA = -98 +/- 19 muA per hemibladder; P less than 0.001, n = 10). The inhibition by T3 was present at 6 nM (P less than 0.01, n = 10) and became not significant at 0.6 nM. T3 had no significant effect on base-line or aldosterone-stimulated H+ transport. Thyroid hormone might therefore regulate the late response of the SCC to aldosterone at the level of its target cell.