Background: As part of a primary prevention of asthma study, we measured the effect of environmental control measures on Der p 1, Fel d 1 and Can f 1 over a 3.5-year period. Methods: High-risk infants (both parents atopic) without pets, were randomized to the Active group (n = 142, vinyl flooring in child's room, allergen-impermeable cot mattress, hot-washable toy, mite allergen-impermeable encasings to parental bed and to child's bed when older, high filtration vacuum cleaner, hot-washing of bedding) or the Control group (n = 136, no intervention), in early pregnancy. Dust samples from the parental mattress, living room floor, child's mattress and floor at baseline (pregnancy), birth and at 3 years were analysed for Der p 1, Fel d 1 and Can f 1. Results: A total of 278 families completed the baseline visit, 259 the birth visit and 239 the 3-year visit. In the Active group at 3 years, 58% remained compliant with all measures likely to reduce the child's exposure to allergen and 77% of parents still used encasings on their bed. Levels of Der p 1, Fel d 1 and Can f 1 were significantly lower in the Active group in the child's floor and the child's mattress at 3 years compared to the Control group (P <0.001). For the parental mattress, the levels of Der p 1 and Fel d 1 were lower in the Active group (P <0.001) and there was a strong trend towards a lower level for Can f 1. There was no difference in the levels of any of the allergens between the groups in the living room floor. Childrens' bedrooms with no detectable mite, cat or dog allergen were significantly more common in the Active than the Control group (25 vs. 2, P <0.001). Conclusions: Environmental control measures are effective in substantially reducing levels of Der p 1, Fel d 1 and Can f 1 in homes without pets in the long term and are acceptable to families. The effect of this environmental manipulation on the development of sensitization and allergic disease remains to be seen.