Flavonoids are plant phenolic compounds involved in leguminous plant-microbe interactions. Genes implied in the central branch (chalcone synthase (CHS), chalcone isomerase (CHI)) or in the isoflavonoid branch of the flavonoid biosynthesis pathway have been characterized in Medicago sativa. No information is available to date, however, on genes whose products are involved in the synthesis of other types of flavonoids. In this paper we present the genomic organization as well as the nucleotide sequence of one flavanone-3-hydroxylase (F3H) encoding gene of M. sativa, containing two introns and exhibiting 82-89% similarity at the amino acid level to other F3H proteins. This is the first report on the genomic organization of a f3h gene so far. We present also the sequence of a partial dihydroflavonol-4-reductase (DFR) M. sativa cDNA clone. Southern blot experiments indicated that f3h and dfr genes are each represented by a single gene within the tetraploid genome of M. sativa. By a combination of Northern blot and RT-PCR analysis, we showed that both f3h and dfr genes are expressed in flowers, nodules and roots, with a pattern distinct from chs expression. Finally, we show that dfr is expressed in M. sativa leaves whereas f3h is not. The role played by these two genes in organs other than flowers remains to be determined.