A combination of Northern blot analysis and in situ hybridization was used to measure and localize changes in the levels of transcripts encoding five enzymes of phenylpropanoid/flavonoid/isoflavonoid metabolism in Medicago truncatula roots following colonization with the mycorrhizal fungus Glomus versiforme. In colonized roots, elevated levels of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) transcripts were detected specifically in the cortical cells containing arbuscules. This localization was discrete, and elevated levels of transcripts were not observed in adjacent, non-colonized cells. In contrast, isoflavone reductase (IFR) transcripts, which were detected at relatively high levels in the cortical cells of non-colonized roots, were almost undetectable in the colonized cortical cells containing arbuscules and were also lowered in adjacent cells. The expression of chalcone isomerase (CHI) and isoliquiritigenin 2′-O-methyltransferase (ChalOMT) was unaffected by colonization, and the tissue-specific patterns of gene expression were the same as in non-colonized roots. It is concluded that the establishment of the mycorrhizal interaction results in cell type-specific differential expression of genes of phenylpropanoid/flavonoid/isoflavonoid biosynthesis, which may be causally related to arbuscule development.