Agrobacterium-mediated transformation of barley is a valuable tool for determining gene function by over-expression of a gene of interest or by RNAi-based gene silencing. The method is based on the inoculation of immature embryos with Agrobacterium and uses a hygromycin resistance gene to allow selection of transgenic plants. The protocol described leads to average transformation efficiencies of 25 % meaning that large numbers of fertile transgenic plants can be produced.