Recent studies have shown that all three subtypes of alpha2-adrenergic receptor (alpha2-AR) are found in brain. The purpose of this study was to map the subtype localization of the alpha2A- and alpha2B-ARs in brain structures. RNase protection shows that both the alpha2A- and alpha2B-ARs are detectable in cortex, cerebellum, pons-medulla, and hypothalamus. We tested probes derived from the alpha2A- and alpha2B-AR cDNAs on cell lines that express each of the alpha2-AR subtypes to establish the subtype specificity of these probes for in situ hybridization. Then we used the alpha2A- and alpha2B-AR probes for in situ hybridization on sagittal and coronal sections of rat brain. Both alpha2A and alpha2B mRNA were detected throughout the brain. Overall, there appears to be a greater expression of message for alpha2A- than alpha2B-AR in most brain areas, with the exception of the thalamus. Developing these probes for in situ hybridization is an important step for further studies on the exact role of the alpha2-AR subtypes in neurons that modulate cardiovascular function.