As part of a case-controlled study on the relationship between home dampness and respiratory symptoms of children, the concentration of the major allergen of Dermato-phagoides pteronyssinus (Der p I) in floor dust and mattress dust in 516 dwellings in the Netherlands was measured. A checklist, completed by the investigators, was used to obtain information on home and occupant characteristics, which may have an impact on the Der p I concentration in house dust. The geometric mean mite antigen concentrations were 2370 ng Der p I/g floor dust for the living room, 2201 ng Der p I/g floor dust for the bedroom and 5075 ng Der p I/g mattress dust. In 86% of the houses the maximum concentration was higher than 2000 ng Der p I/g dust, that is regarded as representing a risk for genetically predisposed individuals for the development of specific IgE to house dust mite allergen. In 55% of the houses the maximum concentration exceeded 10000 ng Der p I/g dust, regarded as a risk factor for acute attacks of asthma for mite allergic patients. The Der p I concentrations in dust from carpeted floors were six to 14 times higher than in dust from floors with a smooth floor covering. Higher Der p I concentrations in floor dust were also significantly associated with increasing age of the dwelling and of the floor covering, with an increasing number of occupants, and with the absence of floor insulation. For mattress dust, the age of the mattress, the presence of an outer cavity wall and mechanical ventilation were important factors. Older mattresses had higher levels, and mattress dust from bedrooms with solid brick outer walls had higher levels than that from bedrooms with outer cavity walls. Mattresses in homes with continuous mechanical ventilation had almost twice lower levels than mattresses in homes with natural ventilation. There was a tendency towards higher Der p I concentrations in dust in homes with reported or observed signs of dampness. The Der p I concentrations in dust from carpeted bedroom floors and mattresses were positively associated with the average relative humidity in the bedroom over a period of 3–6 weeks in a subset of the homes where relative humidity was measured. Similar results were obtained using the concentrations of Der p I in ng/m2 instead of ng/g dust. The results obtained in this study are of importance for planning and evaluating allergen avoidance measures advised to mite allergic patients.