The subcellular localization of the two acyltransferases, tigloyl-CoA: (−)-13α-hydroxymultiflorine/(+)-13α-hydroxylupanine O-tigloyltransferase (HMT/HLTase) and p-coumaroyl-CoA: (+)-epilupinine O- p-coumaroyltransferase (ECTase), for quinolizidine alkaloid biosynthesis has been investigated using the isolated organelles from the hypocotyls of Lupinus albus and L. hirsutus green seedlings. The two acyltransferases were not located in chloroplasts, where de novo synthesis of the quinolizidine alkaloid skeleton is supposed to take place. The HMT/HLTase activity was associated with the mitochondrial fractions and localized in the matrix, judged by the marker enzyme assay. However, the ECTase activity was not associated with the mitochondrial fractions. Intracellular transport of quinolizidine alkaloids is discussed in relation to the biosynthetic pathway of ester alkaloids in Lupinus plants.