The spinosyns are a new genre of fermentation-derived molecules that contain a 12-membered macrocyclic lactone in a unique tetracyclic ring. More than 20 spinosyns (A-Y) have been identified and result from variations in substitution patterns on the two sugars (forosamine and 2',3',4'-tri-O-methylrhamnose) and the tetracyclic ring system. Changes in the substitution pattern on the forosamine nitrogen have little effect on the activity of the spinosyns to neonate tobacco budworm larvae, while loss of a methyl group from the 2', 3' or 4' positions of the 2',3',4'-tri-O-methylrhamnose results in at least a 10-fold reduction in biological activity. Loss of a methyl group at C16 or C21 also reduces activity to neonate tobacco budworm larvae, while addition of a methyl group at C6 either has little effect or may slightly improve activity. The most active spinosyn identified to date to neonate tobacco budworm larvae is spinosyn A, which is also the principal component of spinosad (Tracer^®), a naturally occurring mixture spinosyn A and spinosyn D.