Estrogen receptor alpha (ERalpha) exerts regulatory actions through genomic mechanisms. In the classical pathway, ligand-activated ERalpha binds directly to DNA through estrogen response elements (ERE) located in the promoter of target genes. ERalpha can also exert indirect regulation of transcription via protein-protein interaction with other transcription factors such as AP-1.S everal ERalpha synonymous polymorphisms have been identified and efforts to understand their implications have been made. Nevertheless effects of synonymous polymorphisms are still neglected. This chapter focuses on the experimental procedure employed in order to characterize the transcriptional activity of a synonymous polymorphism of the ERalpha (rs746432) called Alanine 87 (Ala87). Activity of both WT and Ala87 ERalpha isoforms on transcriptional pathways can be analyzed in transiently transfected cells using different reporter constructs. ERalpha efficiency on the classical genomic pathway can be analyzed by determining its transactivation activity on an ERE-driven thymidine kinase (TK) promoter controlling the expression of the luciferase reporter gene. Transcriptional activity through the indirect genomic pathway can be analyzed by employing an AP-1 DNA response element-driven promoter also controlling the expression of luciferase reporter gene.