A fragment (alpha-13) of Staphylococcal alpha-toxin was compared with intact alpha-toxin as regards biochemical and biological properties, and the resulting information was used for mapping biologically-active regions of alpha-toxin. The alpha-13 fragment had an apparent Mr of 18,500, judged by sodium dodecylsulphate polyacrylamide gel electrophoresis. However, it showed the same relative mobility as alpha-toxin when subjected to gel filtration on Biogel P60, high pressure liquid chromatography or electrophoresis on polyacrylamide gradient gel. The fragment had roughly the same specific hemolytic activity as intact alpha-toxin. In contrast to intact alpha-toxin, the fragment was neither membrane-damaging to mouse adrenocortical (Y 1) tumour cells nor lethal to mice. However, a short treatment of Y 1 cells with the fragment completely blocked intoxication by subsequently-added alpha-toxin. Likewise, the lethal effect of alpha-toxin was inhibited when the fragment was injected prior to the toxin. Thus, the fragment had lost the active region(s) responsible for Y 1 cell intoxication and lethality, while the region(s) for binding to these targets, as well as the region responsible for hemolysis, were retained. On the basis of these findings and previous reports concerning tryptic fragments of alpha-toxin, a hypothetical map of the different biologically-active regions of alpha-toxin was established.