Sesquiterpene lactones are a class of natural compounds well-known for their bioactivity and are characteristic for the Asteraceae family. Most sesquiterpene lactones are considered derivatives of germacrene A acid (GAA). GAA can be stereospecifically hydroxylated by the cytochrome P450 enzymes (CYP) Lactuca sativa costunolide synthase CYP71BL2 (LsCOS) and Helianthus annuus GAA 8β-hydroxylase CYP71BL1 (HaG8H) at C6 (in α-orientation) or C8 (in β-orientation), respectively. Spontaneous subsequent lactonization of the resulting 6α-hydroxy-GAA leads to costunolide, whereas 8β-hydroxy-GAA has not yet been reported to cyclize to a sesquiterpene lactone. Sunflower and related species of the Heliantheae tribe contain sesquiterpene lactones mainly derived from inunolide (7,8-cis lactone) and eupatolide (8β-hydroxy-costunolide) precursors. However, the mechanism of 7,8-cis lactonization in general, and the 6,7-trans lactone formation in the sunflower tribe, remain elusive. Here, we show that, in plant cells, heterologous expression of CYP71BL1 leads to the formation of inunolide. Using a phylogenetic analysis of enzymes from the CYP71 family involved in sesquiterpenoid metabolism, we identified the CYP71DD6 gene, which was able to catalyze the 6,7-trans lactonization in sunflowers, using as a substrate 8β-hydroxy-GAA. Consequently, CYP71DD6 resulted in the synthesis of eupatolide, thus called HaES ( Helianthus annuus eupatolide synthase). Thus, our study shows the entry point for the biosynthesis of two distinct types of sesquiterpene lactones in sunflowers: the 6,7-trans lactones derived from eupatolide and the 7,8-cis lactones derived from inunolide. The implications for tissue-specific localization, based on expression studies, are discussed.