The action of L-triiodothyronine (T3) on amino acid transport in the GC clonal strain of rat pituitary cells was investigated by measurement of the uptake of the nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB). The uptake of AIB by GC cells appeared to require energy and Na+ and displayed Michaelis-Menten kinetics. In comparison to cultures maintained in the absence of T3, T3 addition resulted in an increase in AIB uptake which seemed due to an increase in the initial rate of AIB transport. T3 addition resulted in increased AIB accumulation at later time points as well. T3 induction of AIB transport did not occur until 3.5 h after addition of T3, and this effect was blocked by cycloheximide. Maximal induction occurred 48 to 72 h later. One-half maximal induction occurred 24 to 48 h after addition of T3. No detectable changes either in AIB uptake or intracellular water space, measured by uptake of the nonmetabolizable sugar, 3-O-methyl-D-glucose, were noted for the first 120 min after addition of T3. Induction of AIB transport occurred at 0.05 nM T3 (total medium concentration) and one-half maximal induction occurred at 0.17 nM T3. The relative potencies of four iodothyronine analogues for AIB transport were in accord with their reported activities in nuclear T3 receptor binding assays. These data suggest that induction of AIB transport by T3 may be mediated by the nuclear T3 receptor and may reflect the pleiotrophic response of GC cells to thyroid hormone.