This article describes an efficient procedure to study Nod factor-induced gene expression in root hairs of the model legume Medicago truncatula. By developing an improved method of fracturing frozen root hairs, it has been possible to obtain a highly purified root hair fraction from M. truncatula seedlings yielding sufficient RNA for real-time quantitative RT-PCR expression analysis. After Nod factor treatment it was possible to detect up to 100-fold increases of MtENOD11 and pMtENOD11-gus transcript levels in root hair RNA. This corresponds to 5-7-fold higher induction levels than for entire root tissue preparations. Furthermore, the use of these enriched RNA samples has revealed for the first time a very significant induction (30-fold) of the MtENOD40 gene in root hairs in response to Nod factors. It is concluded that the rapid and convenient procedure described here will be particularly useful for detecting tissue-specific low-level gene expression in root hairs responding to Rhizobium Nod factors or other exogenous signals.