To understand further the role of the dynamic interplay between keratinocytes and stromal components in the regulation of the growth, differentiation, morphogenesis, and basement membrane assembly of human stratified squamous epithelium, we have generated novel, three-dimensional organotypic cultures in which skin keratinocytes were grown in the absence or presence of pre-existing basement membrane components and/or dermal fibroblasts. We found that keratinocytes cultured in the presence of pre-existing basement membrane components and dermal fibroblasts for 9 d showed rapid assembly of basement membrane, as seen by a nearly complete lamina densa, hemidesmosomes, and the polarized, linear distribution of laminin 5 and a6 integrin subunit. Basement membrane assembly was somewhat delayed in the absence of dermal fibroblasts, but did occur at discrete nucleation sites when pre-existing basement membrane components were present. No basement membrane developed in the absence of pre-existing basement membrane components, even in the presence of dermal fibroblasts. Bromodeoxyuridine incorporation studies showed that early keratinocyte growth was independent of mesenchymal support, but by 14 d, both fibroblasts and assembled basement membrane were required to sustain growth. Normalization of keratinocyte differentiation was independent of both dermal fibroblasts and structured basement membrane. These results indicated that epithelial and mesenchymal components play a coordinated role in the generation of structured basement membrane and in the regulation of normalized epithelial growth and tissue architecture in an in vitro model of human skin.