This paper reports the detection and identification of two 3',4'-dioxygenated isoflavones from red clover: 3',7-dihydroxy-4'-methoxyisoflavone (calycosin) (1) and 7-hydroxy-3',4'-methylenedioxyisoflavone (pseudobaptigenin) (2). Both isoflavones have previously been reported from a variety of other sources but neither has yet been detected in pasture legumes. Pseudobaptigenin (2) was detected as a minor component (ca 5%) in a bulk crystalline preparation of formononetin (3); no separation of (2) and (3) was achieved by TLC or PC, and the mixture was resolved only by GLC, with the identity of (2) confirmed by GC-MS of the TMSi derivatives compared with an authentic sample. Calycosin (1) was isolated from the mother liquor and crystallised; PMR in DMSO-d6 indicated a pattern characteristic of 7,3',4'-hydroxylation and it was distinguished from a previously reported isomeric form by a positive Gibbs test. Isoflavones (1) and (2) were also detected in ether extracts of freeze-dried plant material not exposed to acid or alkali during extraction, so they are unlikely to be artefacts of plant preparation or isolation. The detection of (1) and (2) with ortho oxygenation in ring B is of considerable biosynthetic interest. Dewick's feeding experiments showed formononetin (3) was incorporated into pterocarpans maackiain (10) and medicarpin (11) in CuCl₂-treated red clover seedlings, but 2'-hydroxyformononetin (6) and its corresponding isoflavanone (8) were incorporated into (11) only, implying formation of the methylenedioxy moiety of (10) must precede 2'-hydroxylation. Our results show red clover contains methylenedioxyisoflavonoid (2) lacking a 2'-hydroxyl group, and (1) has been suggested as a likely precursor of (2). The co-occurrence of (1) and (2) with (10) in red clover, along with earlier reports of their co-occurrence in other species, suggests a biosynthetic pathway for (10) in this plant: (3) → (1) → (2) → (7) → (9) → (10). Initial steps in the biosynthesis of the corresponding coumestan medicagol (12) are likely to be similar.