The uptake of myo-inositol by mouse embryonic stem (ES) cells was measured using [2-3H] myo-inositol. Uptake of myo-inositol by ES cells occurred in a mainly saturable, sodium-, time- and temperature- dependent manner, which was inhibited by glucose, phloridzin and ouabain. Self inhibition by inositol was much greater than inhibition by glucose indicating that transport was not occurring via a sodium- dependent glucose transporter. Uptake rate was much greater than efflux rate indicating a mainly unidirectional transport mechanism. Estimated kinetics parameters for sodium-dependent inositol uptake were a K-m of 65.1 +/- 11.8 mu mol L-1 and a V-max of 5.0 +/- 0.59 pmol mu g protein(-1) h(-1). Inositol uptake was also sensitive to osmolality; uptake increased in response to incubation in hypertonic medium indicating a possible role for inositol as an osmolyte in ES cells. These characteristics indicate that myo-inositol transport in mouse ES cells occurs by a sodium-dependent myo-inositol transporter protein.