Vacuoles were isolated by osmotic rupture of mesophyll protoplasts from the primary leaves of 4-dand 7-d-old plants of rye (Secale cereale L.). Their content of two flavones, luteolin 7-O-[13-o-glucuronosyl-(1 ~2)13-D-glucuronide] (R2) and luteolin 7-O-[13-D-glucuronosyl (1 ~2)13-D-glucuronide]-4'-O-13-D-glucuronide (Ra) , as well as that of three specific flavone-glucuronosyltransferases involved in their biosynthesis and of a specific ~-glucuronidase was determined in comparison to the parent protoplasts. The two flavonoids were found to be entirely located in the vacuolar fraction, together with 70% of the activity of UDP-glucuronate: luteolin 7-O-diglucuronide-4'-O-glucuronosyl-transferase (LDT; EC 2.4.1.), the third enzyme of the sequence of three transferases in the anabolic pathway. The activities of the first and second anabolic enzymes, UDP-glucuronate: luteolin 7-O-glucuronosyltransferase (LGT; EC 2.4.1.) and UDP-glucuronate: luteolin 7-O-glucuronide-glucuronosyltransferase (LMT; EC 2.4.1.) could not be found in the vacuolar fraction in appreciable amounts. The specific 13-glucuronidase (EC 3.2.1.), catalyzing the deglucuronidation of luteolin triglucuronide to luteolin diglucuronide, was present with 90% of its activity in the digestion medium after isolation of mesophyll protoplasts, indicating an apoplastic localization of this enzyme. The data presented indicate a directed anabolic and subsequent catabolic pathway for the luteolin glucuronides in the mesophyll cells of rye primary leaves. This includes two cytosolic and a last vacuolar step of glucuronidation of luteolin, and the vacuolar storage of the luteolin triglucuronide. We propose the transport of the latter into the cell wall, after which the * Dedicated to Professor Ludwig Bergmann, Botanisches Institut der Universit~it zu K61n, on the occasion of his 65th birthday ** To whom correspondence should be addressed Abbreviations: LDT=UDP-glucuronate: luteolin 7-O-di-glucuronide-4'-O-glucuronosyltransferase; LGT = UDP-glucuronate: luteolin 7-O-glucuronosyltransferase; LMT=UDP-glucuronate: luteolin 7-O-glucuronide-glucuronosyltransferase triglucuronide is deglucuronidated, this being the first step for further turnover.