Abstract: Squamous cell carcinomas (SCC) represent a substantial clinical problem because of increases, frequent recurrences and successive de novo tumors, especially in organ transplant recipients. To improve upon the current surgical and other non-selective therapies, a validated organotypic in vitro model of primary human SCC needs to be developed. Such a model will have obvious advantages over current cell line and animal based approaches, and may render the latter partly obsolete. In a first approach, an explant technique of primary SCC biopsies onto dermal constructs was used to emulate tumor expansion in an in vitro model. Histological analysis revealed the formation of nests of squamous cells, mimicking an invasive morphological feature of primary SCC. Immunohistochemical analysis comprised an array of markers characteristic of keratinocyte (hyper) proliferation (K6, K16, K17 and Ki67), differentiation (K1, K10 and involucrin), basement membrane (collagen types IV and VII, integrins α6 and β4 and laminin 332) and SCC (K4, K13 and Axl). The generated human SCC models displayed disturbed differentiation and keratins associated with hyperproliferation, but a low frequency of Ki67 positive cells. Basement membrane composition of the in vitro SCC model resembled that of normal skin. These results show for the first time that in vitro modelling of three-dimensional growth of primary cutaneous human SCC is feasible. This model may provide a platform to develop refined preventive and curative treatments and thereby gain understanding of SCC pathogenesis.