In this study, we identified five novel polymorphisms in the estrogen receptor beta (ERbeta) gene in an African population. Interestingly, two of these variants are expected to change the amino acid sequence of the ERbeta protein. These changes correspond to an isoleucine to valine substitution at amino acid position 3 (I3V) and a valine to glycine substitution at position 320 (V320G), respectively. The functional consequences of these amino acid substitutions were determined in different in vitro assays. The I3V mutation displayed no differences with regard to transcriptional activity in a reporter assay, as compared with the wild-type receptor. The V320G mutation, however, showed significantly decreased maximal transcriptional activity in a reporter assay, although its binding affinity for 17beta-estradiol was not affected. A pull-down assay indicated that the interaction of full-length TIF2 with hERbetaV320G was weaker than with hERbetawt. Moreover, surface plasmon resonance analysis revealed reduced interaction of the V320G ERbeta variant with the NR box I and II modules of TIF2. To our knowledge, this represents the first identification of a functional polymorphism in the ERbeta gene. This novel polymorphism provides a tool for human genetic studies of diseases in the African population.