SpoIISAB is a toxin–antitoxin module encoded on the chromosomes of Bacillus subtilis and related Bacilli species. The SpoIISA toxin was previously shown to target the cytoplasmic membrane and to induce lysis in both B. subtilis and E. coli, however the precise manner of SpoIISA toxicity remains unknown. In this work we focused on the N-terminal, transmembrane domain of SpoIISA and verified the prediction of its topology. Using truncated SpoIISA constructs we show that the entire transmembrane domain is required for its toxicity. Moreover, we propose that the oligomerization of this transmembrane domain is crucial for activity of SpoIISA, possibly by forming a pore-like structure.This article is protected by copyright. All rights reserved.