Spores of Bacillus subtilis were exposed to a series of stratosphere simulations. In total, five distinct treatments measured the effect of reduced pressure, low temperature, high desiccation, and intense ultraviolet (UV) irradiation on stratosphereisolated and ground-isolated B. subtilis strains. Environmental conditions were based on springtime data from a mid-latitude region of the lower stratosphere (20 km). Experimentally, each treatment consisted of the following independent or combined conditions: -70 °C, 56 mb, 10-12%relative humidity and 0.00421, 5.11, and 54.64 W/m² of UVC (200-280 nm), UVB (280-315 nm), UVA (315-400 nm), respectively. Bacteria were deposited on metal coupon surfaces in monolayers of ~1 x 10⁶ spores and prepared with palagonite (particle size< 20 μm). After 6 h of exposure to the stratosphere environment, 99.9% of B. subtilis spores were killed due to UV irradiation. In contrast, temperature, desiccation, and pressure simulations without UV had no effect on spore viability up through 96 h. There were no differences in survival between the stratosphere-isolated versus ground-isolated B. subtilis strains. Inactivation of most bacteria in our simulation indicates that the stratosphere can be a critical barrier to long-distance microbial dispersal and that survival in the upper atmosphere may be constrained by UV irradiation.