Germinating alfalfa (Medicago sativa L.) seeds release flavonoids and betaines that induce transcription of nodulation (nod) genes in Rhizobium meliloti and promote growth of R. meliloti and mycorrhizal fungi. Defining how these beneficial signal molecules vary among alfalfa cultivars and other Medicago species is one step toward improving agronomic management of rhizosphere microbes. This project quantified flavonoids and betaines released from seeds of seven Medicago species and measured their biological activities in R. meliloti. Seed compounds were separated by HPLC, identified by spectroscopic methods, and quantified by comparison with authentic standards. Regulatory activities of seed rinse fractions were measured as NodD1- or NodD2-dependent transcription of a nodC-lacZ fusion in R. meliloti. The betaines trigonelline and stachydrine were released from all species examined (M. sativa, M. falcata M. littoralis, M. polymorpha, M. orbicularis, M. ruthenica, and M. truncatula), and the amount of trigonelline correlated positively (r(2) = 0.60; P< 0.05) with NodD2-dependent nod-gene induction. Luteolin and luteolin-7-O-glucoside flavonoids were released from all species except M. littoralis, M. ruthenica, and M. truncatula. NodD1-dependent nod-gene induction correlated positively (r(2) = 0.74; P < 0.05) with the total amount of those compounds. Exudation kinetics of the flavonoid nod-gene inducer chrysoeriol in alfalfa differed greatly from other flavonoids and may reflect a metabolic event rather than simple solubilization. Variation in the amounts of flavonoids released from the limited number of alfalfa cultivars tested suggested that plant selection techniques may increase these beneficial, but sometimes limiting, compounds.