Mitogen-activated protein kinases (MAPKs) appear to be ubiquitously involved in signal transduction during eukaryotic responses to extracellular stimuli. In plants, no heat shock-activated MAPK has so far been reported. Also, whereas cold activates specific plant MAPKs such as alfalfa SAMK, mechanisms of such activation are unknown. Here, we report a heat shock-activated MAPK (HAMK) immunologically related to ERK (Extracellular signal-Regulated Kinase) superfamily of protein kinases. Molecular mechanisms of heat-activation of HAMK and cold-activation of SAMK were investigated. We show that cold-activation of SAMK requires membrane rigidification, whereas heat-activation of HAMK occurs through membrane fluidization. The temperature stress- and membrane structure-dependent activation of both SAMK and HAMK is mimicked at 25°C by destabilizers of microfilaments and microtubules, latrunculin B and oryzalin, respectively; but is blocked by jasplakinolide, a stabilizer of actin microfilaments. Activation of SAMK or HAMK by temperature, chemically modulated membrane fluidity, or by cytoskeleton destabilizers is inhibited by blocking the influx of extracellular calcium. Activation of SAMK or HAMK is also prevented by an antagonist of calcium-dependent protein kinases (CDPKs). In summary, our data indicate that cold and heat are sensed by structural changes in the plasma membrane that translates the signal via cytoskeleton, Ca2+ fluxes and CDPKs into the activation of distinct MAPK cascades.