The majority (85%) of 394 monarch butterflies sampled from overwintering sites in Mexico contain the same epoxy cardenolide glycosides, including most conspicuously a novel polar glycoside with a single genin-sugar bridge (aspecioside), as occur in the milkweedsAsclepias speciosa andA. syriaca. This cardenolide commonality was established by isolating aspecioside and syriobioside from the wings of overwintering monarchs and the two plant species, and comparing Chromatographie and NMR spectrometric characteristics of the isolates. When combined with the migratory pattern of monarchs and the distribution of these two milkweed species, this chemical evidence lends strong support to the hypothesis thatA. syriaca is the major late summer food plant of monarchs in eastern North America. This finding may be of ecological importance, forA. syriaca contributes less cardenolide and cardenolides of lower emetic potency to monarchs than most milkweeds studied to date.