Novel, functional skin staining with fluorescent, ultradeformable lipid vesicles (Transfersomes™, IDEA, Munich, Germany) was developed and combined with confocal laser scanning microscopy. This revealed the structural and barrier characteristics of intact skin to a resolution of ≥ 0.2 μm, that is, to the limit of light microscopy. Different routes of penetration into the stratum corneum were visualized and new details in the skin anatomy and barrier were unveiled. Most prominent was the lateral inhomogeneity of the stratum corneum, where three to 10 neighbouring corneocyte 'columns' were found to form a cluster. Corneocyte edges inside each cluster intercalated extensively, but adjacent clusters were separated by 'gorges' a few micrometres deep; lipid packing was also less regular and tight in the intercluster region. Two quantitatively different hydrophilic pathways were found in the horny layer: an intercluster route with low penetration resistance comprising ≤ 1% of the total or ≤ 20% of the pathway area in the skin, and an intercorneocyte pathway that resists penetration better and is more abundant (≥ 3% of the skin or ≥ 80% of the pathway area). This latter route is strongly tortuous, as it goes between all the corneocytes in a cluster. It traces the irregularities between the intercellular lipid lamellae and/or the adjacent corneocyte envelopes which may act as virtual channels in the skin. It was inferred that such channels coincide with the route of water evaporation through the skin and exhibit the permeability barrier maximum in the stratum corneum conjunctum.