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<p>Due to low concentrations and chemical complexity, in situ observations of bioaerosol are geographically and temporally sparse, and this limits the accuracy of current emissions inventories. In this study, we apply a new methodology, including corrections for misidentification of mineral dust, to measurements of single particles over four airborne sampling campaigns to derive vertical profiles of bioaerosol over the continental United States. The new methodology is based on single-particle mass spectrometry (SPMS); it can extend historic datasets to include measurements of bioaerosol, it allows comparisons to other techniques, and it generally agrees with a global aerosol model. In the locations sampled, bioaerosols were at least a factor of 10 less abundant than mineral dust. Below 2&thinsp;km, bioaerosol concentrations were measured between <span class="inline-formula">6×10³</span> and <span class="inline-formula">2×10⁴</span>&thinsp;m<span class="inline-formula">⁻³</span>. Between 2 and 8&thinsp;km, bioaerosol concentrations were between 0 and <span class="inline-formula">2×10⁴</span>&thinsp;m<span class="inline-formula">⁻³</span>, and above 8&thinsp;km, bioaerosol concentrations were between 0 and <span class="inline-formula">1×10³</span>&thinsp;m<span class="inline-formula">⁻³</span>. Between 30&thinsp;% and 80&thinsp;% of single bioaerosol particles detected were internally mixed with dust. A direct comparison of the SPMS methodology with a co-located wideband integrated bioaerosol sensor (WIB) fluorescence sensor on a mountaintop site showed agreement to within a factor of 3 over the common size range.</p>