Rhizobia excrete variously substituted lipo-oligosaccharide Nod factors into the legume rhizosphere. Homologous legumes respond to these signals through deformation of the root hairs and the development of nodulation foci in the root cortex. Cellular events in root hairs from the susceptible zone of nearly mature root hairs were studied in root segments loaded with the calcium indicators Fura-2 or Fluo-3. Application of 10⁻⁹ M Nod factors of the broad-host-range Rhizobium sp. NGR234 to the homologous legume Vigna unguiculata resulted, within seconds, in plateau-like increases in intracellular free calcium ([Ca²⁺]i) in the root hairs and root epidermal cells. Nod factors of R. meliloti at 10⁻⁹ M caused equally rapid increases in [Ca²⁺]i in the root hairs and epidermal cells of the nonhost V. unguiculata, and also induced root-hair deformation. The chitin tetramer, N-N′-N″-N′″-tetracetylchitotetraose, which represents the backbone of Nod factors, induced neither root-hair deformation nor changes in [Ca²⁺]i in V. unguiculata. Root hairs and epidermal cells of the nonlegume non-host Arabidopsis thaliana showed neither [Ca²⁺]i increases nor root-hair deformation in response to both factors.