For many agents, the aerodynamic particle size can affect both the virulence and disease course in animal models. Botulinum neurotoxins (BoNTs), which are widely known as potential bioterrorism agents, have been shown to be toxic via multiple routes of exposure, including small particle inhalation (1-3 µm MMAD). However, the impact of larger particle sizes on the potency of BoNT has not been previously reported. In this study, we compared the potency of BoNT in small and large particle aerosols. Outbred mice (ICR (CD-1 ® )) were exposed to BoNT-containing aerosols with differing mass median aerodynamic diameters (MMADs) of 1.1, 4.9, and 7.6 microns. The effects of bioaerosol sampler and inhalation exposure modality were studied. Collecting aerosolized BoNT onto gelatin filters or into liquid impingers resulted in equivalent estimates of aerosol concentration. Nose-only and whole-body inhalation exposure resulted in nearly identical estimates of the median lethal dose (LD 50 ). The LD 50 for inhaled BoNT increased approximately 50-fold when the median aerodynamic particle size was increased from 1.1 to 4.9 µm, from 139 (95% CI: 111-185) to 7324 (95% CI: 4287-10 891) mouse intraperitoneal median lethal doses (MIPLD 50 ). These results demonstrate the importance of aerodynamic particle size and regional deposition patterns with regards to BoNT inhalational toxicity. These data will be useful for medical countermeasure development, as well as biodefense preparedness modeling by demonstrating that the estimates of dose and toxicity of an inhaled aerosol containing BoNT can be significantly affected by a range of factors.