Sawfly larvae of the tribe Phymatocerini (Hymenoptera: Tenthredinidae), which are specialized on toxic plants in the orders Liliales and Ranunculales, exude a droplet of deterrent hemolymph upon attack by a predator. We investigated whether secondary plant metabolites from Ranunculaceae leaves are sequestered by phymatocerine Monophadnus species, i.e., Monophadnus alpicola feeding upon Pulsatilla alpina and Monophadnus monticola feeding upon Ranunculus lanuginosus. Moreover, two undescribed Monophadnus species were studied: species A collected from Helleborusfoetidus and species B collected from Helleborus viridis. Comparative high-performance liquid chromatographicphotodiode array detection-electrospray ionization-mass spectrometric analyses of plant leaf and insect hemolymph extracts revealed the presence of furostanol saponins in all samples. Larvae of species A and B actively sequestered (25R)-26-[(alpha-L-rhamnopyranosyl) oxy]-22alpha-methoxyfurost-5-en-3beta-yl O-beta-D-glucopyranosyl-(1–3)-O-[6-acetyl-beta-D-glucopyranosyl-(1–3)]-O-beta-D-glucopyranoside (compound 1). This compound occurred at a 65- to 200-fold higher concentration in the hemolymph of the two species (1.6 and 17.5 micromol/g FW, respectively) than in their host plant (0.008 and 0.268 micromol/g FW, respectively). In M. monticola, compound 1 was found at a concentration (1.2 micromol/g FW) similar to that in the host plant (1.36 micromol/g FW). The compound could not be detected consistently in M. alpicola larvae where, however, a related saponin may be present. Additional furostanol saponins were found in H. foetidus and H. viridis, but not in the two Monophadnus species feeding on them, indicating that sequestration of compound 1 is a highly specific process. In laboratory bioassays, crude hemolymph of three Monophadnus species showed a significant feeding deterrent activity against a potential predator, Myrmica rubra ant workers. Isolated furostanol saponins were also active against the ants, at a concentration range similar to that found in the hemolymph. Thus, these compounds seem to play a major role for chemical defense of Monophadnus larvae, although other plant secondary metabolites (glycosylated ecdysteroids) were also detected in their hemolymph. Physiological and ecological implications of the sequestered furostanol saponins are discussed.