Thymocytes obtained from suckling or young adult rats were used as a model system to study the action of thyroid hormones in vitro. In this tissue, L-triiodothyronine (T3) increased the uptake of the non-metabolizable amino acids, alpha-aminoisobutyrate and cycloleucine. A detectable effect of T3 on the uptake of cycloleucine was seen at a concentration of 0.1 muM and maximum effects were seen at 20 muM. Thyroxine (T4) also increased cycloleucine uptake with about one-third the potency of T3, and this effect could not be ascribed to conversion of T4 to T3. In contrast, L-monoidotyrosine and L-diiodotyrosine were without effects on transport. Kinetic studies indicated that T3 enhanced uptake by inhibiting amino acid efflux; no effect was seen on influx. The effect of T3 on amino acid uptake was evident within 1 min, and was not inhibited by either prior treatment of the cells with cycloheximide or by lowering the incubation temperature from 37 to 24 C. In other studies, when T3 was injected into rats in vivo at a dose of 20 mug/100 g, the uptake of cycloleucine was enhanced in thymocytes obtained 1 h later. These data suggest that thyroid hormones can directly influence amino acid transport in rat thymocytes. This effect is prompt, is independent of new protein synthesis, and may reflect a direct interaction with specific components of the cell membrane.