The behavior of the gonadotropin-releasing hormones (GnRH) neurons controlling fertility is dependent upon cyclic fluctuations in circulating concentrations of estrogen. However, the nature of estrogen action upon these cells has remained controversial due to their dispersed distribution within the brain, and evidence indicating that they do not express nuclear estrogen receptors (ERs) in vivo. We report here an acute brain slice preparation that enables individual living GnRH neurons to be identified within the mouse brain and show, using single cell multiplex RT-PCR, that the greater than 50% of GnRH neurons in adult and prepubertal females contain ERalpha messenger RNA. Approximately 10% of GnRH neurons contained ERbeta transcripts that were always coexistent with ERalpha. Single cell RT-PCR analysis of nonGnRH cells located in the medial preoptic area revealed a similar coexpression pattern of ERalpha and ERbeta transcripts. In contrast, single striatal cells were not found to contain ERbeta despite ERalpha being present in approximately 25% of cells. The analysis of single GnRH neurons in cycling female mice revealed that the detection of ERalpha and ERbeta transcripts was lowest on proestrus (ERalpha, 18% of all GnRH neurons; ERbeta, 0%) compared with diestrus (44% and 6%) and estrus (75% and 19%, respectively). Using a novel approach that enables single cell RT-PCR analysis of GnRH neurons, we present here evidence for the cyclic expression of ERalpha and ERbeta messenger RNAs within prepubertal and adult female GnRH neurons.