Immunofluorescence and immuno-electron microscopy have been used to test the hypothesis that flavonoid metabolism is organized as a membrane-associated enzyme complex. The cellular and subcellular locations of chalcone synthase (CHS) and chalcone isomerase (CHI), the first two enzymes of this pathway, were examined in Arabidopsis roots. High levels of both enzymes were found in the epidermal and cortex cells of the elongation zone and the root tip, consistent with the accumulation of flavonoid endproducts at these sites. Co-localization of CHS and CHI was observed at the endoplasmic reticulum and tonoplast in these cells, and also in electron-dense regions that are, as yet, unidentified. In addition, a striking asymmetric distribution was observed for these enzymes in cortex cells of the elongation zone, which may provide clues about the physiological function of flavonoids in roots. The accumulation of CHS and CHI was also examined in tt7(88), a mutant in the gene for flavonoid 3'-hydroxylase (F3'H), which has been postulated to serve as a membrane anchor for the flavonoid enzyme complex. CHS and CHI accumulated to lower levels in cortex cells and higher levels in epidermal cells in the roots of this mutant as compared with wild-type plants. Moreover, the electron-dense regions containing these two enzymes were not observed. However, localization of CHS and CHI to the ER and tonoplast did not appear to be affected, suggesting that other proteins may function in recruiting the "soluble" flavonoid enzymes to membranes. Staining of flavonoid endproducts with DPBA was consistent with expression of CHS and CHI in these seedlings.