The p53-regulated 14-3-3sigma gene encodes an inhibitor of cell cycle progression essential for senescence and clonal evolution of keratinocytes in vitro. Here we analysed the in vivo expression of 14-3-3sigma protein in several skin diseases, which are characterized by hyperproliferative keratinocytes. Unexpectedly, the 14-3-3sigma protein was expressed at high levels in psoriasis (11 of 11 patients), condylomata acuminata (11/11), actinic keratoses (11/11) and squamous cell carcinomas (SCC) (11/11). However, keratinocytes that had undergone transformation to basal cell carcinoma (BCC) showed partial (10 of 41; 24.4%) or complete (19 of 41; 46.3%) loss of 14-3-3sigma protein expression. BCC (5/5), SCC (6/6) and actinic keratoses (7/7) concomitantly expressed the p53-homolog p63 and 14-3-3sigma at high levels, ruling out potential inhibitory effects of p63 isoforms on 14-3-3sigma transcription as the basis for loss of 14-3-3sigma expression. Of 41 BCC samples isolated by laser-capture microdissection, 28 (68.3%) showed CpG-hypermethylation of the 14-3-3sigma promoter combined with reduced or absent 14-3-3sigma protein levels in 22 cases (78.6%). Since it has been reported that BCC retain wild-type p16(INK4A) and here BCC with CpG-methylation of 14-3-3sigma did not show CpG-methylation of p16(INK4A) (0/17), silencing of 14-3-3sigma may contribute to evasion of senescence in BCC. As experimental removal of 14-3-3sigma sensitizes to DNA damage, silencing of 14-3-3sigma may explain the high efficacy of radiation therapy in the treatment of BCC.