The biosynthesis of lophocerine (1.2.3.4-tetrahydro-1-isobutyl-6-methoxy-2-methylisoquinolin-7-ol) in intact Lophocereus schottii plants has been studied. The C5 unit of the alkaloid probably arises independently from both leucine and mevalonic acid. 3-Methylbut-3-enyl pyrophosphate, 3-methylbutan-1-ol, and 3-methylbutanal have been shown to be intermediates in the biosynthesis. Our work suggested that mevalonic acid (I) was a more efficient precursor of lophocerine than was leucine (V). However too great a reliance could not be placed on this result as the cacti used in the two experiments were not in a comparable state of vigour. We have now repeated these experiments using identical cacti under identical feeding conditions and confirm that mevalonate is incorporated approximately twice as efficiently as leucine into lophocerine. Although this result suggests a dual origin for the C5 fragment of lophocerine, the possibility exists that leucine is metabolised to mevalonic acid, by an established pathway, and so incorporated into the alkaloid. To test this hypothesis it was decided to feed 3-methylbutanoic acid, which, in the form of its coenzyme A ester, is reported to be an intermediate on the pathway from leucine to mevalonic acid, to the cactus. The lophocerine was inactive, suggesting that leucine is not incorporated via mevalonic acid. In separate experiments 3-methyl[1-14C]but-3-enyl pyrophosphate (II), 3-methyl[1-14C]butan-1-ol (III), and 3-methyl[1-14C]butanal (IV) were each injected into two L. schottii cacti. The cacti were grown on for 14 days and pure active lophocerine was isolated as described previously. Specific activities and percentage incorporations are reported in the Table. The lophocerine from the 3-methylbutanal feed was degraded to 3,4-dimethoxyphthalic anhydride (VII) which had the same specific activity as the lophocerine, showing that the aldehyde was incorporated specifically into the alkaloid. The comparatively low but definite specific incorporation of 3-methylbutanal suggests that this compound is the intermediate involved in the ring closure reaction. The incorporations into lophocereine of the pyrophosphate (II) and the alcohol (III) support the biosynthetic pathway shown in the Scheme.