The effects of the Ca²⁺ channel blockers verapamil, nifedipine, and diltiazem on triiodothyronine (T₃) and thyroxine (T₄) uptake were tested in cultured cardiomyocytes from 2-day-old rats. Experiments were performed at 37°C in medium with 0.5% BSA for [¹²⁵I]T₃ (100 pM) or 0.1% BSA for [¹²⁵I]T₄ (350 pM). The 15-min uptake of [¹²⁵I]T₃ was 0.124 ± 0.013 fmol/pM free T₃ (n = 6); [¹²⁵I]T₄ uptake was 0.032 ± 0.003 fmol/pM free T₄ (n = 12). Neither T₃ nor T₄ uptake was affected by 1% DMSO (diluent for nifedipine and verapamil). Uptake of [¹²⁵I]T₃ but not of [¹²⁵I]T₄ was dose dependently reduced by incubation with 1-100 µM verapamil (49-87%, P < 0.05) or nifedipine (53-81%, P < 0.05). The relative decline in [¹²⁵I]T₃ uptake after 4 h of incubation with 10 µM verapamil or nifedipine was less than after 15 min or 1 h, indicating that the major inhibitory effect of the Ca²⁺ channel blockers occurred at the level of the plasma membrane. The reduction of nuclear [¹²⁵I]T₃ binding by 10 µM verapamil or nifedipine was proportional to the reduction of cellular [¹²⁵I]T₃ uptake. Diltiazem (1-100 µM) had no dose-dependent effect on [¹²⁵I]T₃ uptake but reduced [¹²⁵I]T₄ uptake by 45% (P < 0.05) at each concentration tested. Neither the presence of 20 mM K⁺ nor the presence of low Ca²⁺ in the medium affected [¹²⁵I]T₃ uptake. In conclusion, the inhibitory effects of Ca²⁺ channel blockers on T₃ uptake in cardiomyocytes are not secondary to their effects on Ca²⁺ influx but, rather, reflect interference with the putative T₃ carrier in the plasma membrane.