Hirsutellin (HtA) is intermediate in size between other ribotoxins and less specific microbial RNases, and thus offers a unique chance to determine the minimal structural requirements for activities unique to ribotoxins. Here, we have determined the structure of HtA by NMR methods. The structure consists of one alpha-helix, a helical turn and seven beta-strands that form an N-terminal hairpin and an anti-parallel beta-sheet, with a characteristic alpha + beta fold and a highly positive charged surface. Compared to its larger homolog alpha-sarcin, the N-terminal hairpin is shorter and less positively charged. The secondary structure elements are connected by large loops with root mean square deviation (rmsd) values > 1 A, suggesting some degree of intrinsically dynamic behavior. The active site architecture of HtA is unique among ribotoxins. Compared to alpha-sarcin, HtA has an aspartate group, D40, replacing a tyrosine, and the aromatic ring of F126, located in the leucine 'environment' close to the catalytic H113 in a similar arrangement to that found in RNase T1. This unique active site structure is discussed in terms of its novel electrostatic interactions to understand the efficient cytotoxic activity of HtA. The contributions of the N-terminal hairpin, loop 2 and loop 5 with regard to protein functionality, protein-protein and protein-ipid interactions, are also discussed. The truncation and reduced charge of the N-terminal hairpin in HtA may be compensated for by the extension and new orientation of its loop 5. This novel orientation of loop 5 re-establishes a positive charge on the side of the molecule that has been shown to be important for intermolecular interactions in ribotoxins.