The NEPPK are a heterogenous group of disorders characterised by thickening of palms and soles due to abnormal keratinization. We studies two families with focal NEPPK which was inherited as an autosomal dominant with an age of onset of 6-7 years. Keratosis pilaris was seen on the arms and legs with a whitish opalescence over the hard palate, buccal mucosa and glans penis of circumcised males. Immunofluorescence was performed with the light and confocal microscope with specific monoclonal antibodies to keratin polypeptides on skin biopsies from the palms, buccal mucosa and non-involved skin from affected individuals. Similar biopsies were taken from 5 healthy controls. The K6 and K16 staining pattern were abnormal with reduced K6 and K16 expression in the palmoplantar epidermis; aberrant K16 expression in the arm and increased in the buccal mucosa. Expression of suprabasal K1, K10 and the palmoplantar specific K9 was normal in all sites. Segregation analysis with markers flanking the keratin gene clusters excluded 12q as the site of mutation and demonstrated linkage to 17q to a locus at 17q12-q21 (Z=3.2 at q=0.0), however the size of pedigrees meant that meaningful lod scores could only be obtained for one family. Here, we describe mutations in the keratin 16 gene in the two families with NEPPK. A mutation was found in the helix initiation motif of K16 (R129S) in a affected member in one family. Two affected members of the other focal family show a mutation in the same region of K16 (N127S). Mutations were excluded from the control population. These mutations in K16 gene are the first to be described in focal NEPPK and may be implicated in the pathogenesis of this disease.