To determine the size distribution of the spores of Bacillus anthracis, and compare its size with other Bacillus species grown and sporulated under similar conditions. Spores from several Bacillus species, including seven strains of B. anthracis and six close neighbours, were prepared and studied using identical media, protocols and instruments. Here, we report the spore length and diameter distributions, as determined by transmission electron microscopy (TEM). We calculated the aspect ratio and volume of each spore. All the studied strains of B. anthracis had similar diameter (mean range between 0.81 +/- 0.08 mu m and 0.86 +/- 0.08 mu m). The mean lengths of the spores from different B. anthracis strains fell into two significantly different groups: one with mean spore lengths 1.26 +/- 0.13 mu m or shorter, and another group of strains with mean spore lengths between 1.49 and 1.67 mu m. The strains of B. anthracis that were significantly shorter also sporulated with higher yield at relatively lower temperature. The grouping of B. anthracis strains by size and sporulation temperature did not correlate with their respective virulence. The spores of Bacillus subtilis and Bacillus atrophaeus (previously named Bacillus globigii), two commonly used simulants of B. anthracis, were considerably smaller in length, diameter and volume than all the B. anthracis spores studied. Although rarely used as simulants, the spores of Bacillus cereus and Bacillus thuringiensis had dimensions similar to those of B. anthracis. Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefence against B. anthracis. The data presented here should help in the selection of simulants that better resemble the properties of B. anthracis, and thus, more accurately represent the performance of collectors, detectors and other countermeasures against this threat agent.