House dust mites have been considered the most important source of allergens for humans. These allergens have been encountered at different indoor sites, mainly on mattresses and pillows. To evaluate the number and different specimens of mites on Brazilian bunk-bed mattresses. Dust samples were collected once using a standardized method on the upper mattress surface (US) and lower mattress surface (LS) (bed frame also included in the latter) of 58 mattresses. The total number of mite bodies on the LS was 3.5-fold higher than on the US, with a mean concentration of 932 mites per gram of fine dust (mites/g) on the US (range, 0-3,375 mites/g) and 3,254 mites/g on the LS (range, 125-14,500 mites/g) (P < .001). Additionally, the number of house dust mite bodies on the LS was 2.4 higher than on the US (P < .001); the mean concentration was 750 mites/g on the US (range, 0-2,875 mites/g) and 1,816 mites/g on the LS (range, 0-10,875 mites/g). Approximately 91% (n = 52) of the US and all LS dust samples had more than the limit of 100 mites/g. The most frequent family was Pyroglyphidae in both mattress surfaces, with Dermatophagoides pteronyssinus the most important species found. Storage mites, including Glycyphagidae (P < .001), Acaridae (P < .001), and other families (P < .001), also showed significant differences in the number of mites between both sample counts. This study shows a significant difference in US and LS mite counts, with higher counts on the LS. Mite allergen avoidance should include the LS and bed frame as potential targets.91559-62