The relationship between the transport of the thyroid hormone T3 and the transport of neutral amino acids was investigated in JAR human placental choriocarcinoma cells. The uptake of leucine, mediated by the amino acid transport system L, was inhibited by T3 and T4, and the nature of inhibition was competitive. Uptake of T3 into the cells was predominantly Na+ independent, and so was that of leucine. However, although an acidic extracellular pH stimulated leucine uptake, the uptake of T3 remained unaffected. In addition, leucine failed to inhibit T3 uptake. The aromatic amino acids phenylalanine and tryptophan were found to inhibit the uptake of T3, but these two amino acids were transported into the cells predominantly via system L. The amino acid transport system T, which is specific for aromatic amino acids, was not detectable in these cells. Treatment of the cells with the calmodulin antagonist CGS 9343 B stimulated the uptake of leucine and tryptophan, but inhibited the uptake of T3. Kinetic analysis of T3 uptake revealed the presence of a single saturable system for this hormone in these cells, and the Michaelis-Menten constant for this system was 0.77 +/- 0.06 microM. Metabolic poisons that interfere with the cellular generation of ATP had no effect on the uptake of T3. It is concluded that in placental choriocarcinoma cells, 1) T3 and T4 are high affinity competitive inhibitors of the amino acid transport system L, 2) uptake of T3 occurs via a specific Na(+)-independent, energy-independent, and saturable mechanism that is unrelated to the amino acid transport systems L and T, 3) the aromatic amino acids phenylalanine and tryptophan interact, although weakly, with the T3 uptake system, and 4) calmodulin-dependent processes participate in the regulation of the T3 uptake system.