Pore-forming cytolytic proteins distributed in a wide variety of eukaryotic and prokaryotic organisms have been intensively studied in terms of pathophysiological functions and molecular architecture of transmembrane pores. These proteins are also being developed for various analytical applications such as detector of proteins and DNA by engineering the structure of the pore. Staphylococcal gamma-hemolysin (Hlg), a pore-forming protein, which consists of two separate proteins, LukF and Hlg2, has potential to be a useful tool as a multifunctional biosensor. However, the fine structure of the Hlg pore has not been clarified. Our previous studies revealed that LukF and Hlg2 assemble alternately on the membrane in a molar ratio of 3:4 and 4:3 and form cylindrical heteroheptameric transmembrane pores. In the present study, we conducted quantitative analysis of the subunit arrangement of the pore by using two-dimensional (2-D) image analysis based on high-resolution transmission electron microscopy (TEM) images. Results of this study suggest a new aspect of the characteristic structure in two-component pore-forming protein and can contribute to the engineering of the Hlg pore.