The crystal structure of beta-purothionin (beta-PT) has been determined at 1.7 A resolution. beta-PT and previously solved alpha(l)-PT belong to a family of membrane-active plant toxins homologous to crambin. (beta-PT crystallizes in the same space group as alpha(l)-PT (1422) but with the c axis 3 A longer than (alpha(l)-PT. The unit-cell dimensions of beta-PT crystals are a = b = 53.94 and c = 72.75 A. Two data sets were collected on a multiwire area detector, each with R(sym) around 6.0%, and were merged to get a single data set at 1.7 A, (R(merge) = 9.6%). The X-ray structure of alpha(l)-PT was used to build a starting model for beta-PT. The beta-PT model was refined using the program PROLSQ from 10 to 1.7 A resolution to an R-factor of 19.8% with very good geometry. The final structure contains 439 atoms including 337 protein atoms, 77 waters, two acetates, two glycerols and one phosphate. The high-resolution structure of the beta-PT agreed well with that of the lower resolution alpha(l)-PT structure only after the latter was extensively rerefined. Both refinements revealed phosphate and glycerol molecules which are important in lattice formation. The binding of phosphate and glycerol molecules to purothionins (PT) was confirmed by NMR and was implicated in the biological activity of toxins. Modeling of phospholipid binding to PT based on glycerol and phosphate-binding site could shed light on the lytic toxicity of this protein-toxin family. Although the structures of (alpha(l)-PT and beta-PT preserve the overall fold of crambin, they exhibit key differences that are directly relevant to the toxicity of thionins.