Cry1Aa, a toxin of Bacillus thuringiensis, is a widely used biological pesticide that has also been considered as a genetically-engineered intrinsic crop protection. It has been shown that the toxin forms cation-selective pores in the host membrane inducing cytolytic activity. Because crystallization of the active (pore) conformation of most pore-forming toxins has not yet been possible, one key problem for understanding the mechanism of these proteins is to determine the number of toxins required to form a single pore and to establish whether the pores have a fixed stoichiometry. Thus, the present study aims to investigate the oligomerization of Cry1Aa in a lipid bilayer using a subunit counting technique based on single molecule fluorescence spectroscopy. Purified Cry1Aa toxins labelled at different positions within the pore-forming domain were inserted into supported lipid bilayer at various concentrations within the single-molecule observation range. Photobleaching steps of single fluorophores in the fluorescence time traces were counted in order to determine the number of subunits for each oligomer. We found that the oligomerization occurs in the membrane and that the distribution of oligomers was best fitted by a binomial distribution of fourth order, indicating a tetrameric pore-forming entity.