The osmotically inducible uptake of betaine (NNN-trimethylglycine) by SV-3T3 cells has been studied and compared with the similar process in MDCK cells. Betaine uptake by SV-3T3 cells could be described in terms of a saturable, Na(+)-dependent, component plus a small non-saturable, Na(+)-independent, component. Transport was active, producing considerable accumulation of betaine in the cells. After exposure of the cells to hypertonic conditions for 6 h, there was a marked increase in betaine uptake. Kinetic analysis indicated that this increase resulted from an increase in the Vmax. value of the saturable component, from about 88 to 185 nmol of betaine/5 min per mg of protein, the corresponding Km values of about 15 and 10 mM not being significantly different. This induction of transport activity was detectable only after about 2 h exposure of the cells to hypertonic medium, closely paralleling an induction of influx of N-methylaminoisobutyric acid, and was prevented by the presence of cycloheximide. Betaine influx was markedly inhibited by several neutral amino acids, particularly those transported by system A, such as N-methylaminoisobutyric acid and the imino acid proline. A high concentration (25 mM) of betaine also significantly inhibited the uptake of proline by SV-3T3 cells. Although very similar results were obtained with MDCK cells, prolonged exposure of cells to hypertonic conditions revealed distinct differences. When the hypertonic incubation was extended from 6 h to 24 h, betaine transport in SV-3T3 cells either remained the same or decreased, whereas it showed a further marked increase in MDCK cells, and also became sensitive to inhibition by gamma-aminobutyric acid. mRNA for the betaine transporter BGT-1 [Yamauchi, Uchida, Kwon, Preston, Brooks Robey, Garcia-Perez, Burg and Handler (1992) J. Biol. Chem. 267, 649-652] was detectable in MDCK cells exposed to hypertonic medium for 24 h, but not in SV-3T3 cells under any conditions. It is concluded that SV-3T3 cells do not produce a specific inducible transporter analogous to BGT-1, but they can accumulate betaine via the amino acid transport system A.