Barley (Hordeum vulgare L.) plants of two commercial cultivars were transformed with sense and antisense constructs of a chitinase class II gene in order to develop a transformation system for this gene in barley. Transformation of embryos with the two antisense constructs resulted in ten regenerated plants, while no plants could be obtained using the sense construct. The presence of the inserted construct could be confirmed for six of the plants by PCR analysis. This system was used to study the role of class II chitinase in the regulation of mycorrhizal symbiosis. The colonization of two of the antisense transformants by the arbuscular mycorrhizal fungus Glomus intraradices was investigated microscopically and by use of signature fatty acids. The arbuscular incidence increased in transformed barley, and one transformant supported higher extraradical mycelium biomass. It is concluded that antisense transformation of barley could be a useful tool in investigations on the symbiosis between barley and mycorrhizal fungi.