Early studies found estrogen-binding sites in the ER knockout (ERalphaKO) mouse brain, suggesting a splice variant of ERalpha or another ER. The discovery of ERbeta suggested that binding was due to ERbeta, although questions about an ERgamma remained. To test this hypothesis, ERbetaKO mice were generated and crossed with ERalphaKO mice, and ERalpha/betaKO animals were used for in vivo binding studies with [(125)I]estrogen. The results revealed nuclear binding sites in the ERalpha/betaKO hypothalamus and amygdala. As the binding resembled the distribution of ERalpha, we evaluated the presence of ERalpha splicing variants. A nonphysiological splice variant of ERalpha was identified in ERalpha/betaKO brain and uterus, but was absent in wild-type mice. ERalpha immunoreactivity was also detected in regions of ERalpha/betaKO brain where residual binding was seen. To ascertain the functionality of the variant, the regulation of PR was assessed in brain. The results revealed that E2 significantly increased PR expression, an indication that the variant can regulate gene transcription. These data demonstrate the presence and functionality of an ERalpha variant in ERalpha/betaKO brain and suggest that the residual binding and regulation of PR in ERalpha/betaKO brain can be accounted for by the variant.