Absent or defective collagen VII at the dermo-epidermal junction is the hallmark of dystrophic recessive epidermolysis bullosa. Little is known of the alterations of other collagenous and non-collagenous components of the basement membrane; it is likely that their assembly may be disturbed by the lack of collagen VII molecules. The spatial relationship of collagen IV and laminin has been studied, both in bullous and in non-bullous areas. Skin biopsies from five patients affected by severe dystrophic recessive epidermolysis bullosa were rapidly frozen and freeze-dried. Collagen IV and laminin were labelled with specific monoclonal antibodies and FITC- or TRITC-conjugated secondary antibodies. Sections were observed with conventional light/fluorescence microscopy and with confocal laser scanning microscopy. Collagen IV and laminin were not co-localized: the former displayed a split image, being present at the floor and the roof of the blister, while the latter was confined to the roof. Confocal microscopy also allowed three-dimensional (3D) reconstruction of the dermo-epidermal junction from a series of optical sections, with viewing of the reconstructed specimen from a sequence of angles. By this procedure, laminin exhibits an irregular, coarsely granular distribution, both in affected and in apparently non-affected areas, while collagen IV appears as a homogeneous sheet. These results show that freeze-drying is the technique of choice for high-resolution immunofluorescence of skin samples and suggest that in dystrophic recessive epidermolysis bullosa, a complex disruption of the extracellular matrix assembly exists even before blister formation, probably due to the lack of collagen VII.