MicroRNAs (miRNAs) finely tune messenger RNA (mRNA) expression. As the brain is a highly heterogeneous tissue, physiologically relevant miRNA expression profiling greatly benefits from sampling brain regions or nuclei. MiRNA expression profiling from individual samples is also important for investigating potential differences between animals according to their physiological and pathophysiological status. We have punched the arcuate (ARC) and paraventricular (PVN) nuclei from the hypothalamus of seven male Wistar rats and used them to establish a novel method for the characterization of the miRNA expression profile of individual rat brain nuclei. The identity of the ARC and PVN samples was checked for proopiomelanocortin and arginine vasopressin mRNA expression, respectively. Individual cDNA libraries were constructed from purified RNAs between 16 and 26 bases, using barcoded adapters. Libraries were multiplexed and sequenced using Illumina technology to a read depth >10(5). The ARC and PVN profiles displayed similar expression from a set of more than 210 miRNA genes. Expression was high or moderate for about twenty miRNAs that may be used to define a common ARC/PVN prototype profile of male Wistar rats. These miRNAs included seven of the eight genes of the let-7 family, the two miR-7 genes, miR-9 gene and 5' copy of the three miR-30 loci. Our method shows that the ARC and PVN from a single rat are accessible for miRNA digital characterization. This method will allow miRNA transcriptome characterization for any rat brain substructure or nuclei that can be microdissected.