The purpose of this study was to establish seasonal levels of Der p 1, Der f 1 and D. pteronyssinus allergens in mattress and floor dust samples in the homes of 20 mite-allergic asthmatic patients in Cartagena, Colombia. Dust samples were collected using a portable vacuum cleaner once monthly for the same 12 months in all houses. The highest Der p 1 levels in mattress samples were detected in August (geometric mean = 109.49 ng/g) and represented a 2.2-times increase from the lowest level observed in November. Der f 1 was detected in only 3 mattresses, ranging from 90 ng to 1.6 micrograms per gram of dust. The highest levels were detected in February and March. The highest level of D. pteronyssinus allergens in mattress dust samples, quantitated by RAST-inhibition, was 8, 994 AU/g (80% inhibition); the lowest level detected was 150 AU/g (5% inhibition). Allergen levels were significantly higher in mattresses than in floor samples (p < 0.001). Overall, a Spearman rank correlation coefficient of 0.48, p < 0.001, was obtained between Der p 1 and D. pteronyssinus allergens. There was a good correlation between mite allergens and absolute humidity (r = 0.8, p = 0.007). Der p 1, Der f 1 and D. pteronyssinus allergen levels show minimal variations in this tropical environment. Allergens derived from various mite species must be considered when assessing mite allergen exposure in these environments.