Demethylavermectins, which lack methyl groups on the oleandrose moiety, were fed to wild-type "Streptomyces avermitilis," which produces the normal avermectin components, and the bioconversion products were identified. The oleandrose units of the demethylavermectins were not methylated by the wild-type strain, but the C-5 of the macrolide ring was methylated. This demonstrates that methylation of the oleandrose units is not a terminal step in avermectin biosynthesis and that it occurs before attachment of the sugars to the macrolide ring. Avermectin biosynthesis thus differs from that of tylosin and erythromycin, in which methylation of the hexose moieties is a terminal reaction.