Hypocretenolides, a small group of sesquiterpene lactones with an unusual ring structure, are constituents of a small number of species from the Lactuceae tribe (Asteraceae). Three biogenetically closely related 14-hypocretenolides from Leontodon hispidus L. were investigated for a putative anti-inflammatory activity. 14-Hydroxyhypocretenolide-beta-D-glucoside-4'-14"-hydroxyhypocr etenoate significantly exhibited in vivo anti-inflammatory activity in the croton oil-induced mouse ear edema. To obtain first information regarding the molecular targets which might be affected by this constituent, two in vitro bioassays were performed: (i) DNA binding activity of the transcription factor NF-kappa B was evaluated by electrophoretic mobility shift assay (EMSA) using TNF-alpha-activated Jurkat T cells and (ii) nitrite accumulation in cell culture supernatants of LPS-activated RAW 264.7 macrophages was determined as a parameter for inducible nitric oxide synthase (iNOS)-dependent nitric oxide release. In order to gain information about structure-activity relationships, additionally the aglycone 14-hydroxyhypocretenolide and its D-glycoside were investigated in these in vitro systems. 14-Hydroxyhypocretenolide-beta-D-glucoside-4'-14"-hydroxyhypocr etenoate as well as its aglycone exhibited activity in both test systems, whereas the D-glucoside was not or only weakly active.