Hypoosmotic shock induced a transient increase in cytosolic free calcium concentration ([Ca 2+] cyt) and subsequent activation of 50-, 75- and 80-kDa protein kinases in tobacco ( Nicotiana tabacum L.) suspension culture cells. Depletion of external calcium suppressed both the elevation of [Ca 2+] cyt and the activation of protein kinases in response to hypoosmotic shock, indicating that the elevation of [Ca 2+] cyt is prerequisite for the activation of protein kinases. Pharmacological analysis indicated that the hypoosmotic shock-activated protein kinases were activated by phosphorylation, suggesting that the activities of these protein kinases are regulated by putative protein kinases. These results suggest that the hypoosmotic signal is transduced to protein kinase cascades which are triggered by [Ca 2+] cyt elevation.