ABSTRACT Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow in media of up to 3.0 M NaCl and pH 11. This cyanobacterium can synthesize betaine from glycine by three-step methylation using S -adenosylmethionine as a methyl donor. To unveil the mechanism of betaine uptake and efflux in this alkaliphile, we isolated and characterized a betaine transporter. A gene encoding a protein (BetT A. halophytica ) that belongs to the betaine-choline-carnitine transporter (BCCT) family was isolated. Although the predicted isoelectric pH of a typical BCCT family transporter, OpuD of Bacillus subtilis , is basic, 9.54, that of BetT A. halophytica is acidic, 4.58. BetT A. halophytica specifically catalyzed the transport of betaine. Choline, γ-aminobutyric acid, betaine aldehyde, sarcosine, dimethylglycine, and amino acids such as proline did not compete for the uptake of betaine by BetT A. halophytica . Sodium markedly enhanced betaine uptake rates, whereas potassium and other cations showed no effect, suggesting that BetT A. halophytica is a Na + -betaine symporter. Betaine uptake activities of BetT A. halophytica were high at alkaline pH values, with the optimum pH around 9.0. Freshwater Synechococcus cells overexpressing BetT A. halophytica showed NaCl-activated betaine uptake activities with enhanced salt tolerance, allowing growth in seawater supplemented with betaine. Kinetic properties of betaine uptake in Synechococcus cells overexpressing BetT A. halophytica were similar to those in A. halophytica cells. These findings indicate that A. halophytica contains a Na + -betaine symporter that contributes to the salt stress tolerance at alkaline pH. BetT A. halophytica is the first identified transporter for compatible solutes in cyanobacteria.