The nitrogen-fixing symbiosis between Rhizobiaceae and legumes is one of the best-studied interactions established between prokaryotes and eukaryotes. The plant develops root nodules in which the bacteria are housed, and atmospheric nitrogen is fixed into ammonia by the rhizobia and made available to the plant in exchange for carbon compounds. It has been hypothesized that this symbiosis evolved from the more ancient arbuscular mycorrhizal (AM) symbiosis, in which the fungus associates with roots and aids the plant in the absorption of mineral nutrients, particularly phosphate. Support comes from several fronts: 1) legume mutants where Nod⁻ and Myc⁻ co-segregate, and 2) the fact that various early nodulin (ENOD) genes are expressed in legume AM. Both strongly argue for the idea that the signal transduction pathways between the two symbioses are conserved. We have analyzed the responses of four classes of non-nodulating Melilotus alba (white sweetclover) mutants to Glomus intraradices (the mycorrhizal symbiont) to investigate how Nod⁻ mutations affect the establishment of this symbiosis. We also re-examined the root hair responses of the non-nodulating mutants to Sinorhizobium meliloti (the nitrogen-fixing symbiont). Of the four classes, several sweetclover sym mutants are both Nod⁻ and Myc⁻. In an attempt to decipher the relationship between nodulation and mycorrhiza formation, we also performed co-inoculation experiments with mutant rhizobia and Glomus intraradices on Medicago sativa, a close relative of M. alba. Even though sulfated Nod factor was supplied by some of the bacterial mutants, the fungus did not complement symbiotically defective rhizobia for nodulation.