Olive tree is a warm-temperature evergreen tree with low tolerance to frost, although cultivars which differ in terms of cold acclimation have been empirically selected. In herbaceous species, free cytosolic calcium ([Ca 2 + ] c ) is involved in cold acclimation. The objective of this study was to measure [Ca 2 + ] c signalling in the olive tree during cold acclimation and to assess the possibility of using [Ca 2 + ] c as an early genotype-selection marker of cold susceptibility. To this end, non-cold-acclimated and cold-acclimated leaf protoplasts of cultivars differing in terms of cold susceptibility were analysed.Cold shocks of various amplitude applied to non-cold-acclimated protoplasts caused consistent and transient increases in [Ca 2 + ] c . A decrease of 0.05 o C/s (i.e. ΔT/dt=2.5 o C/50s) was the threshold cooling rate at which a significant increase in [Ca 2 + ] c could still be observed. When the protoplasts were incubated with either 8-(N,N-di-methylamino)octyl 3,4,5-trimethoxy-benzoate (TMB-8; organelle Ca 2 + channel blocker) or Gd 3 + (plasma membrane Ca 2 + channel blocker), applying the threshold cooling rate, the increase in [Ca 2 + ] c was partially inhibited, suggesting that both an intracellular release of Ca 2 + and an influx through the plasma membrane are involved. When applying repeated cold shocks, the transient increases in [Ca 2 + ] c were reduced only when using a non-severe ΔT/dt. In protoplasts subjected to standard acclimation, the increases in [Ca 2 + ] c were further reduced, or inhibited, depending on the cold susceptibility of the cultivar, suggesting that the Ca 2 + response is involved in a long-term adaptation to cold.