Hypotonic stress causes rapid cell swelling and initiates various cellular adaptive processes. However, it is unknown how cells initially sense low osmolarity and convert it into intracellular signals. We investigated the signal transduction mechanism initiated by hypotonic cell swelling in cardiac myocytes using c-fos expression as a nuclear marker. Treatment of myocytes with hypotonic culture media rapidly induced c-fos expression, whereas hypertonic stress had no effect. Transfection of c-fos reporter gene constructs suggested that the hypotonic stress response element maps to the serum response element of the c-fos promoter. Hypotonic stress immediately (within 5 s) activated tyrosine kinase activity, while activation of ERK1/2 peaked at 5 min. Stress-activated kinase (JNK1) was modestly activated at 15 min, whereas HOG1 like kinase (p38) was not activated by hypotonic stress. Extensive pharmacological studies indicated that only tyrosine kinase inhibitors suppressed the hypotonic swelling-induced c-fos expression. The effect of hypotonic stress was mimicked by chlorpromazine, which is known to cause membrane deformation. These results suggest that the signaling mechanism of hypotonic stress is distinct from that of hyperosmolar stress in mammalian cells. Tyrosine kinase activation is the earliest detectable cell response and plays an essential role in hypotonic swelling-induced ERK1/2 activation and c-fos expression.