Hyperosmotic (405 mosmol/l) exposure of RAW 264.7 mouse macrophages led to a stimulation of betaine uptake and an increase in betaine transporter (BGT-1) mRNA levels. Conversely, hypoosmotic (205 mosmol/l) exposure decreased betaine uptake and diminished BGT-1 mRNA levels. Betaine uptake was Na(+)-dependent and was inhibited by about 90% by GABA, whereas inhibition by methylaminoisobutyrate and myoinositol was less than 15%. Addition of betaine strongly diminished BGT-1 mRNA levels in cells exposed to normoosmotic or hyperosmotic media. When mouse macrophages were preloaded with betaine, lowering of the extracellular osmolarity was followed by a rapid betaine efflux from the cells. This study identifies a constitutively expressed and osmosensitive betaine transporter in RAW 264.7 macrophages and the use of betaine as an osmolyte in these cells.