Respiratory burst oxidase homologues (RBOHs) of the human phagocyte gp91^phox gene have been isolated from several plant species and the proteins that they encode have been shown to play important roles in the cellular response to biotic stress via the production of superoxide. In this study we have identified and preliminarily characterised six RBOHs from barley (Hordeum vulgare L.). Conservation of the genomic structure and conceptual protein sequence was observed between all six barley RBOH genes when compared with Arabidopsis and rice RBOH gene family members. Four of the six barley RBOH transcripts had wide-spread constitutive spatial expression patterns. The inducible expression profiles of HvRBOHF1 and HvRBOHF2 in response to infection by the necrotrophic fungal pathogens Pyrenophora teres f. teres Drechsler and Rhynchosporium secalis (Oudem) J. Davis were further characterised by quantitative real-time PCR (qPCR). Increased expression of both transcripts was observed in leaf epidermal tissue in response to infection, which is in keeping with a suggested role for both transcripts in the early oxidative burst during the plant response to pathogen invasion. This research provides a basis for further analysis and establishment of the roles of this RBOH family in various reactive oxygen species dependent processes in barley.