An acid rhizosphere may interfere with the transcription of nodulation genes in Rhizobium spp. by modifying the production of legume root exudates, or the rhizobial response to them. Certain annual species of Medicago (M. murex, M. polymorpha) can nodulate better in acid soils and solutions than other species (M. truncatula, M. littoralis). The mechanisms of this acid tolerance in nodulation are poorly understood. Root exudates collected at pH 5.8 and pH 6.0 from acid-tolerant species of annual medics induced the expression of nodulation genes in Rhizobium meliloti as indicated from a lacZ gene fusion, whereas exudates from acid-sensitive species grown at these pH values displayed decreased induction activity. For the acid-sensitive host, M. truncatula, increasing the Ca concentration from 0.5 to 5.0 mM at pH 5.8 increased the nod-gene induction activity of its exudates, but there was no effect at higher pH. There was no effect of Ca or pH on the nod-gene induction activity of exudates collected from M. murex. These results indicate a likely mechanism underlying differences in ability to nodulate under acid stress among annual species of Medicago.