1. The hypothesis that cellular hydration state modulates muscle glycogen synthesis was tested by measuring the incorporation of [14C]glucose into glycogen (glycogen synthesis) in primary rat myotubes after experimentally induced volume changes. 2. Glycogen synthesis in myotubes increased (by 75%, P < 0.01) after swelling induced by 60 min exposure to hyposmotic media (170 mosmol kg-1) relative to isosmotic control (300 mosmol kg-1) values, it decreased (by 31%, P < 0.05) after shrinkage induced by 60 min exposure to hyperosmotic (430 mosmol kg-1) media. Myotube 2-deoxy-D-glucose (0.05 mM) uptake was unaffected by changes in external osmolality. 3. Wortmannin (100 nM; 60 min), a phosphatidylinositol 3-kinase inhibitor, decreased basal glycogen synthesis by 28% whereas rapamycin (100 nM; 60 min), which blocks the activation of p70 S6 kinase, had no effect. Both wortmannin (100 nM; 60 min) and rapamycin (100 nM; 60 min) blocked the changes in glycogen synthesis resulting from hypo- and hyperosmotic exposure. 4. Myotube glycogen synthesis is modulated by volume changes independently of changes in glucose uptake. The phenomenon may be physiologically important in promoting glycogen storage during circumstances of myofibrillar swelling, e.g. after feeding or exercise.