Characterizing the variation in bioaerosol concentrations is important for the estimation of health effects associated with bioaerosols and planning exposure assessment strategies. This investigation was conducted in order to develop a better understanding of exposure to fungal spores, pollen, and (1→3)-β-d-glucan, by determining the variations of their concentrations between and within homes. In the study, 24-h air sampling was performed on five consecutive days in four Cincinnati area homes. The samples (a total of 160) were taken simultaneously in four different rooms inside each home and at four different outside locations near the home using Button Personal Inhalable Aerosol Samplers. The relative sizes of the between- and within-home variability to the total variability were calculated for each outcome. The relative sizes of the between- and within-home variability in indoor air ranged from 0.10 to 0.52 and 0.09 to 0.10, respectively. For outdoor air, the between- and within-home variability ranged from 0.27 to 0.50 and 0.09 to 0.10, respectively. Thus, the ranges of within-home variability, both indoors and outdoors, were much less than the variability between different homes. The results suggest that, if long-term sampling methods are employed to characterize the bioaerosol exposure for a population, the sampling should be repeated in a larger number of homes as an alternative to replicate sampling in a fewer number of homes. When characterizing exposure within one home, the sampling should be repeated in different rooms, rather than repeating it on different days.