Most rove beetles of the genus Stenus protect themselves against microorganisms and predators such as ants and spiders by producing the alkaloid stenusine (1) in their pygidial glands. The biosynthesis of 1 was previously investigated in S. bimaculatus, where L-lysine forms the piperidine ring, L-isoleucine the side chain, and acetate the N-ethyl group. In addition to 1, S. similis keeps the pyridine alkaloid (Z)-3-(2-methyl-1-butenyl)pyridine (2) in its pygidial glands. Feeding S. similis beetles with [D,15N]-labeled amino acids followed by GC/MS analysis showed that L-Lys yields the pyridine ring and L-Ile the 2-methyl-1-butenyl side chain. Thus the alkaloids 1 and 2 probably share two precursor molecules in their biosynthesis.