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In this study, air samples were collected simultaneously at ground level and from a broadcast tower (open space) 238 m high using two BioSamplers and two portable Biostage impactors. The sampling lasted up to 5 h, from morning to afternoon, and the experiments were independently conducted twice. The samples collected by the BioSamplers were analyzed for bacterial diversity and concentration, endotoxin and (1,3)-beta-d-glucan content. Denaturing gradient gel electrophoresis (DGGE) fingerprinting, DNA sequencing, real-time quantitative polymerase chain reaction (qPCR), and Limulus amebocyte lysate (LAL) were used for the analyses. Air samples collected by the portable Biostage impactors were directly incubated at 25A degrees C, and the culturable bacteria and fungi concentrations were manually counted. The results from the culture analysis indicated that the ground air samples had higher (statistically significant) concentrations of culturable bacteria and fungi than those taken at an elevation of 238 m. DGGE profiles of PCR products showed similar bacterial diversity in both ground-level and elevated air samples. Pseudomonas was identified by DNA sequencing as the predominant bacterial genus present. The SYBR qPCR tests revealed that the pooled air samples from both the ground and elevated levels had similar total bacterial concentrations, about 1,000 times their culturable ones. Although the endotoxin and (1,3)-beta-d-glucan concentrations were observed to vary over the time, no statistically significant differences were observed in the pooled air samples from the ground and elevated levels over the course of the day. The results from this study elucidated the microbial differences in air at different elevations, and added to the body of evidence of atmospheric mixing of biological agents.