Rhizobium-made Nod factors induce rapid changes in both Ca2+ and gene expression. Mutations and inhibitors that abolish Nod-factor-induced Ca2+ spiking block gene induction, indicating a specific role for Ca2+ spiking in signal transduction. We used transgenic Medicago truncatula expressing a ‘cameleon’ Ca2+ sensor to assess the relationship between Nod-factor-induced Ca2+ spiking and the activation of downstream gene expression. In contrast to ENOD11 induction, Ca2+ spiking is activated in all root-hair cells and in epidermal or pre-emergent root hairs cells in the root tip region. Furthermore, cortical cells immediately below the epidermal layer also show slow Ca2+ spiking and these cells lack Nod-factor-induced ENOD11 expression. This indicates a specialization in nodulation gene induction downstream of Nod-factor perception and signal transduction. There was a gradient in the frequency of Ca2+ spiking along the root, with younger root-hair cells having a longer period between spikes than older root hairs. Using a Ca2+-pump inhibitor to block Ca2+ spiking at various times after addition of Nod factor, we conclude that about 36 consecutive Ca2+ spikes are sufficient to induce ENOD11–GUS expression in root hairs. To determine if the length of time of Ca2+ spiking or the number of Ca2+ spikes is more critical for Nod-factor-induced ENOD11 expression, jasmonic acid (JA) was added to reduce the rate of Nod-factor-induced Ca2+ spiking. This revealed that even when the period between Ca2+ spikes was extended, an equivalent number of Ca2+ spikes were required for the induction of ENOD11. However, this JA treatment did not affect the spatial patterning of ENOD11–GUS expression suggesting that although a minimal number of Ca2+ spikes are required for Nod-factor-induced gene expression, other factors restrict the expression of ENOD11 to a subset of responding cells.