System A is one of the most highly regulated transport systems for transport of neutral amino acids into mammalian cells. Stimulation of uptake of alpha-[3H]methylaminoisobutyric acid (MeAIB), a nonmetabolizable system A substrate, by a novel insulin-sensitizing agent, troglitazone, in 3T3-L1 adipocytes was investigated. Treating adipocytes with troglitazone alone resulted in a time- and dose-dependent increase in the uptake of MeAIB. The peak stimulation appeared about 24 h after troglitazone addition. Both troglitazone- and insulin-stimulated transport activities increased markedly after the induction of differentiation of preadipocytes into adipocytes, and declined to a steady state level in adipocytes. The stimulated MeAIB uptake exhibited substrate specificity typical of system A and was mediated by a single component as determined by Eadie-Hofstee plots. The stimulation by troglitazone and that by insulin were similarly sensitive to actinomycin D and cycloheximide, suggesting that both agents may induce de novo synthesis of the same type of system A transport. Apart from the insulin-independent effect, troglitazone also showed an insulin-dependent action characterized by enhanced sensitivity to insulin. The synergistic stimulation of MeAIB uptake by coadministration of insulin and troglitazone was most prominent at the early stages of adipocyte differentiation. Pretreating cells with troglitazone during the differentiation attenuated the sensitivity of insulin to inhibition by actinomycin D, suggesting that troglitazone may enhance the insulin action by stabilizing messenger RNA involved in system A function.