In focal PPK, K16 gene mutations have been identified in the highly conserved la region of keratins, but epidermolysis does not occur in vivo. Keratinocytes were cultured from an individual with a keratin 16 mutation (R129S) and were immortalised by lipofection using an HPV16 construct (PJ4Ω16). Following crisis, stabilisation and cloning, a stable keratinocyte cell line (KPP1) was developed. The keratin expression of these cells were characterised by immunocytochemistry using a panel of monospecific antikeratin antibodies in both standard monolayer and organotypical cultures (collagen gels and de-epidermalised dermis grown at air/liquid interface). Keratin extracts were also examined by 1D gel electrophoresis and Western blotting for multiple keratins. K14, K6 and K16 were widely expressed with a filamentous pattern of staining. K7 and K17 were also expressed, with small numbers of cells showing K19 positivity. K1 and K10 were minimally expressed in the differentiated cells at the centre of the colonies, except in organotypical cultures, where a stratified, more differentiated epidermis was reproduced, with substantial K6, K16, K1, K10, involucrin and vimentin expression. The keratin intermediate filaments were organised in a filamentous pattern without aggregation as in vivo. Mutation in the K16 gene was confirmed by RT PCR and sequencing of cDNA from the cell line. This keratinocyte line in organotypical culture provides an excellent in vitro model for functional analysis of K16 mutations and their interactions with other structural proteins.