The amatoxins, highly toxic components of Amanita mushrooms, strongly inhibit the DNA‐dependent RNA polymerase II (or B) in eukaryotic cell nuclei. For optimal binding to the enzyme a γ‐hydroxyisoleu‐cine side chain in the 3‐position is important as in γ‐amanitin (compound l), where the OH‐group is bound in the [S]‐configuration. Amanullin, a non‐toxic component, having an oxygen‐free isoleucine side chain no. 3, exhibits an inhibitory effect on RNA polymerase II about two orders of magnitude smaller than that of γ‐amanitin. An equal, relatively weak, inhibitory effect has previously been found with the synthetically obtained Ile3‐analog 7. In the present paper the synthesis of an analog (2) bearing a γ‐hydroxyl group in the isoleucine side chain is described. The compound was found to have about the same inhibitory effect on RNA polymerase II fromDrosophilaembryos as amanullin and the Ile3‐analog 7. Structure analysis by X‐ray diffraction revealed that the hydroxyl group at the ‐carbon atom of side chain‐3 has the [R]‐configuration, the new analog thus being ‐deoxo[()‐hydroxy‐[Ile3]‐amaninamide. It follows that the [S]‐configuration of this chiral center is a prerequisite to maximal toxicity. Crystallographic data demonstrating great similarity between the peptide backbones of the new