Transepidermal water loss (TEWL) is a measure of the steady-state water vapour flux crossing the skin to the external environment and it has been used extensively to characterise skin barrier function. We have previously hypothesised that in vivo TEWL is directly related to the reciprocal of the diffusional permeation pathlength through the stratum corneum (SC). The aim of the present paper is to validate experimentally this hypothesis. Ninety volunteers were recruited and TEWL and corneocyte surface areas were measured for six anatomic sites. The number of cell layers in the SC was calculated for each anatomic site in order to estimate the geometric pathlength for water efflux. Significant anatomic site variability was found for both TEWL and corneocyte surface area which were inversely correlated. A direct reciprocal relationship between TEWL and pathlength was determined, with TEWL values tending to zero when corneocytes are infinitely large. In general, skin sites with smaller corneocytes have fewer cell layers, with shorter permeation pathlengths and higher TEWL values. The results confirm our previous hypothesis and suggest that TEWL may be used to characterise the permeation routes for different anatomic sites.