In some tumors, phosphatase and tensin homolog (PTEN) inactivation may have prognostic importance and predictive value for targeted therapies. Immunohistochemistry (IHC) may be an effective method to demonstrate PTEN loss. It was claimed that PTEN IHC showed poor reproducibility, lack of standardization, and variable effects of preanalytical factors. In this study, we developed an optimal protocol for PTEN IHC, with clone 6H2.1, by checking the relevance of analytical variables in normal tissue and tumors of endometrium, breast, and prostate. Pattern and intensity of cellular staining and background nonspecific staining were quantified and subjected to statistical analysis by linear mixed models. The proposed protocol showed a statistically best performance (P .001). However, there was a trend of significance for decreased staining and fixation under high temperature. Moreover, staining was better in endometrial aspirates than in matched hysterectomy specimens, subjected to less controlled preanalytical variables (mean histoscores, 80 and 40, respectively; P = .002). A scoring system combining intensity of staining and percentage of positive cells was statistically associated with PTEN alterations (P = .01). ; The study was done according to the research collaboration with Dako Denmark A/S. The research team was also supported by grants FIS PI100922, Fundación Mutua Madrileña AP75732010, 2009SGR794, RD12/0036/0013, Fundación Asociación Española contra el Cancer, programa de intensificación de la investigación, Instituto Carlos III, Verelst Baarmoederkankerfonds, Leuven, and European Network for Individualized Treatment of Endometrial Carcinoma. F. A. is senior researcher for the research fund Flandersb. Tumor samples were obtained with the support of XarxaCatalana de Bancs de Tumors, the TumorBanc Platform of RTICC, and Red de Biobancos (RD09/0076/00059)