In the rhizosphere plants meet a wide array of microorganisms. In favorable interactions, such as arbuscular mycorrhizal (AM) and nitrogen fixing symbioses, a dialogue is progressively established between the two interacting organisms to make the appropriate partner choice. These two-way communications rely on the interchange of signals released by both potential symbionts. After perception of the signalling molecules, a signal transduction pathway is induced, leading to the activation of the proper genetic and developmental program in both partners.Variations in intracellular free Ca 2+ concentration occur as one of the initial steps in signalling pathways activated in plants when they encounter pathogens, 1 fungal biocontrol agents 2 and nitrogen-fixing bacteria. 3 Molecules secreted by microorganisms, after binding to specific receptors, trigger in plant cells transient changes in cytosolic Ca 2+ level, due to the influx of the ion from the extracellular environment and/or the release from internal Ca 2+ storage compartments. 4,5 Ca 2+ messages delivered to plant cells are at least partly deciphered on the basis of their spatial and temporal features. The occurrence of different Ca 2+ signatures guarantees the specificity of the ensuing physiological responses.In the legume-rhizobium symbiosis a definite pattern of Ca 2+ oscillations has been reported to occur in response to the rhizobial signalling molecule, the Nod factor, in the nucleus and perinuclear cytoplasm of the root hair. 6 The Ca 2+ spike number has been recently demonstrated to regulate nodulation gene expression. 7 Legumes are able to engage in a dual symbiotic interaction, with rhizobia and AM fungi. Components of the Ca 2+ -mediated signalling pathway are shared by the two symbioses. 8 In the mycorrhizal signal transduction pathway the involvement of Ca 2+ has long been speculated, based on the observed similarities with symbiotic nitrogen fixation. 3 To evaluate the possible participation of Ca 2+ in the early steps of the AM symbiosis, we have used a simplified experimental system given by plant cell suspension cultures stably expressing the bioluminescent Ca 2+ -sensitive reporter aequorin. 9 The use of cultured cells circumvents the problem posed by multilayered organs: in aequorin-transformed seedlings, possible Ca 2+ changes occurring in rhizodermal cells-the first place where the AM fungal signals are perceived and transduced-can be misrecorded due to luminescence calibration over all root cell layers, resulting in an underestimation of the Ca 2+ signal in the responsive cells. An experimental design based on challenging host plant cells with the culture medium of different AM fungi (Gigaspora margarita, Glomus mosseae and intraradices) provided the first firm evidence that Ca 2+ is involved as intracellular messenger during mycorrhizal signalling, at least in a pre-contact stage. Cytosolic Ca 2+ changes, characterized by specific kinetic parameters, were triggered by diffusates obtained from AM resting and germinating spores, 9 and ext...