Phosphatidylcholine (PC) production is accelerated by glucocorticoid, such as dexamethasone (DEX), which enhances fetal lung maturation, promotes differentiation of alveolar type II (ATII) cells, and increases production of both lipid and protein components of lung surfactant. We previously demonstrated that inhibition of choline uptake by ATII cells leads to a decrease of PC synthesis. Since choline uptake may play a critical role in PC production and lung surfactant homeostasis for normal breathing, it is of interest to characterize transporters controlling the disposition of choline in ATII cells. Therefore, we studied the gene regulation and activity of choline transporters in A549 cells, a human ATII cell line. A549 cells were exposed to DEX for 24 h, and mRNA expression levels of choline transporters-like protein 1, (CTL1) and CTL2, were measured using real-time reverse transcription polymerase chain reaction. CTL1 and CTL2 mRNAs were strongly induced by DEX treatment of A549 cells, and the DEX-treated cells showed a significant increase in initial uptake rate of [(3)H]choline, which was assessed under ATP-depleted conditions to block the influence of consumption of choline by choline kinase. Transfection of A549 cells with either CTL1- or CTL2-small interfering RNAs significantly decreased [(3)H]choline uptake. In conclusion, choline transport in A549 cells is increased by treatment with DEX, and the increase is mediated by induction of functional choline transporters CTL1 and CTL2.