The potential health benefits of dietary isoflavones have generated considerable interest in engineering the synthesis of these phytoestrogens into plants. Genistein glucoside production (up to 50 nmol g(-1) fresh weight) was engineered in alfalfa (Medicago sativa) leaves by constitutive expression of isoflavone synthase from Medicago truncatula (MtIFS1). Glucosides of biochanin A (4'-O-methylgenistein) and pratensein (3'-hydroxybiochanin A) also accumulated. Although MtIFS1 was highly expressed in all organs examined, genistein accumulation was limited to leaves. MtIFS1-expressing lines accumulated several additional isoflavones, including formononetin and daidzein, in response to UV-B or Phoma medicaginis, whereas the chalcone and flavanone precursors of these compounds accumulated in control lines. Enhanced accumulation of the phytoalexin medicarpin was observed in P. medicaginis-infected leaves of MtIFS1-expressing plants. Microarray profiling indicated that MtIFS1 expression does not significantly alter global gene expression in the leaves. Our results highlight some of the challenges associated with metabolic engineering of plant natural products, including tissue-specific accumulation, potential for further modification by endogenous enzyme activities (hydroxylation, methylation, and glycosylation), and the differential response of engineered plants to environmental factors.