Imprinting control regions (ICRs) are domains within imprinted loci that are essential for their establishment and maintenance. Imprinted loci can extend over several megabases, encompass both maternally and paternally-expressed genes and exhibit multiple and complex epigenetic modifications including large regions of allele-specific DNA methylation. Differential chromatin organisation has also been observed within imprinted loci but is restricted to the ICRs. In this study we report the identification of a novel imprinting control region for the mouse Neuronatin gene. This biochemically defined putative ICR, present within its 250bp second intron, functions as transcriptional activator in Drosophila. This is unlike other known ICRs which have been shown to function as transcriptional silencers. Furthermore, at the endogenous locus, the activating signal from the ICR extends to the Neuronatin promoter via allele-specific unidirectional nucleosomal positioning. Our results support the proposal that the Neuronatin locus employs the most basic mechanism for establishing allele-specific gene expression and could provide the foundation for the multiplex arrangements reported at more complex loci.