Until recently, synonymous mutations (which do not change amino acids) have been much neglected. Some evidence suggests that this kind of mutations could affect mRNA secondary structure or stability, translation kinetics and protein structure. To explore deeper the role of synonymous mutations, we studied their consequence on the functional activity of the estrogen receptor alpha (ERalpha). The ERalpha is a ligand-inducible transcription factor that orchestrates pleiotropic cellular effects, at both genomic and non-genomic levels in response to estrogens. In this work we analyzed in transient transfection experiments, the activity of ERalpha carrying the synonymous mutation Ala87, a polymorphism involving about 5-10% of the population. In comparison to the wild type receptor, our results show that ERalphaA87 mutation reduces the transactivation efficiency of ERalpha on an ERE reporter gene while its expression level remains similar. This mutation enhances 4-OHT-induced transactivation of ERalpha on an AP1 reporter gene. Finally, the mutation affects the subcellular localization of ERalpha in a cell type specific manner. It enhances the cytoplasmic location of ERalpha without significant changes in non-genomic effects of E2. The functional alteration of the ERalphaA87 determined in this work highlights the relevance of synonymous mutations for biomedical and pharmacological points of view.