Four species of Ithomiinae butterflies (Lepidoptera: Nymphalidae) were observed in nature taking up pyrrolizidine alkaloids (PAs) from withered flowers of Prestonia amabilis J. F. Morales (Apocynaceae, Echiteae) in a montane rainforest in southern Ecuador (ca. 1000 m a.s.l.). Quantitative experiments were subsequently carried out using either withered flowers or crushed roots of P. amabilis. Field trials were conducted in November 2000 at six locations in the area of the Reserva Biológica San Francisco (1800–2000 m a.s.l., ca. 15 km from the first site) where P. amabilis was not known to occur. A total of 40 specimens of 10 species of the Ithomiinae butterflies and 40 specimens of eight species of Arctiidae moths were quantitatively sampled. While the first group showed a clear preference for baits with withered flowers, the latter preferred the crushed roots. In total, 13 species of Ithomiinae were observed visiting PA sources. Eight of these 13 species have no previous records of pharmacophagy for PAs. Within the Ithomiinae, there is evidence of phylogenetic difference in attraction, with a noticeably high proportion of recorded species belonging to the Napeogenini. Analysis of plant material by gas chromatography–mass spectrometry revealed the presence of two novel retronecine monoesters of the lycopsamine type, tentatively identified as 5′-demethyllycopsamine (ideamine A) and 5′-demethylisolycopsamine or one of its diastereoisomers. The highest PA levels were found in flowers of P. amabilis (0.13%, dry weight basis), followed by roots (0.075%) and leaves (0.044%). In flowers, ideamine A accounted for 84% of total PAs, whereas roots contained ideamine A (53%) and 32% of its 3′-acetyl ester. We suggest that Prestonia R. Br. may have served as an ancestral source of PAs in the evolution of pharmacophagous behavior in the Ithomiinae.