Changes in intracellular calcium in pea root hairs responding to Rhizobium leguminosarum bv. viciae nodulation (Nod) factors were analyzed by using a microinjected calcium-sensitive fluorescent dye (dextran-linked Oregon Green). Within 1-2 min after Nod-factor addition, there was usually an increase in fluorescence, followed about 10 min later by spikes in fluorescence occurring at a rate of about one spike per minute. These spikes, corresponding to an increase in calcium of Ϸ200 nM, were localized around the nuclear region, and they were similar in terms of lag and period to those induced by Nod factors in alfalfa. Calcium responses were analyzed in nonnodulating pea mutants, representing seven loci that affect early stages of the symbiosis. Mutations affecting three loci (sym8, sym10, and sym19) abolished Nod-factor-induced calcium spiking, whereas a normal response was seen in peas carrying alleles of sym2 A , sym7, sym9, and sym30. Chitin oligomers of four or five N-acetylglucosamine residues could also induce calcium spiking, although the response was qualitatively different from that induced by Nod factors; a rapid increase in intracellular calcium was not observed, the period between spikes was lower, and the response was not as sustained. The chitin-oligomer-induced calcium spiking did not occur in nodulation mutants (sym8, sym10, and sym19) that were defective for Nod-factor-induced spiking, suggesting that this response is related to nodulation signaling. From our data and previous observations on the lack of mycorrhizal infection in some of the sym mutants, we propose a model for the potential order of pea nodulation genes in nodulation and mycorrhizal signaling.A nitrogen-fixing root nodule is the culmination of a developmental program that begins as a sequence of signal exchanges between plant and bacterial symbionts. In leguminous plants, the first step of this process is rhizobial perception of plant metabolites (typically flavonoids) present in the rhizosphere (1). This stimulates bacterial synthesis and secretion of signaling molecules called Nod factors (2, 3), which are lipochitin oligosaccharides usually containing four or five -1,4-linked N-acetylglucosamine residues with an N-acyl group at the nonreducing end (2). Modifications made to this basic structure are a major factor in determining which legume species will be nodulated by a given rhizobial strain. Thus, for example, the Nod factors made by Sinorhizobium meliloti (which nodulates alfalfa) differ from those of Rhizobium leguminosarum bv. viciae (which nodulates peas and vetch) in the length and unsaturation of their acyl chains and have an O-linked sulfate group at the reducing end (4, 5). R. leguminosarum bv. viciae produces a mixture of four Nod factors in which a tetrameric or pentameric sugar backbone carries a C 18:1 or C 18:4 N-linked acyl group and an O-acetyl group on the acylated sugar (5).Nod factors induce several characteristic biochemical, genetic, and morphological responses when recognized by the appropriate legume (6...