To investigate apoplastic responses of barley (Hordeum vulgare L.) to the barley powdery mildew fungus Blumeria graminis f. sp. hordei, noninvasive microprobe techniques were employed. H(+)- and Ca(2+)-selective microprobes were inserted into open stomata of barley leaves inoculated with Blumeria graminis f. sp. hordei race A6 conidia. Resistance gene-mediated responses of barley genotype Ingrid (susceptible parent line) and the near-isogenic resistant Ingrid backcross lines (I-mlo5, I-Mla12, and I-Mlg) were continuously monitored from 20 min to 4 days after inoculation. The main events were categorized as short-term responses around 2 h after inoculation (hai), intermediate responses around 8 and 12 hai, and long-term responses starting between 21 and 24 hai. Short-term responses were rapid transient decreases of apoplastic H(+)- and Ca2+ activities that lasted minutes only. Kinetics were similar for all genotypes tested, and thus, these short-term responses were attributed as nonspecific first encounters of fungal surface material with the host plasma membrane. This is supported by the observation that a microinjected chitin oligomer (GlcNAc)8 yielded similar apoplastic alkalinization. Intermediate responses are trains of H+ (increase) spikes that, being different in susceptible Ingrid and penetration-resistant I-mlo5 (or I-Mlg), were interpreted as accompanying specific events of papillae formation. Long-term events were massive slow and long-lasting alkalinizations up to two pH units above control. Since these latter changes were only observed with near-isogenic hypersensitive reaction (HR)-mounting genotypes I-Mla12 and I-Mlg but not with I-mlo5 or, to a smaller extent, with susceptible Ingrid, both lacking significant rates of HR, they were rated as cell death specific. It is concluded that apoplastic pH changes are important indicators of host-pathogen interactions that correlate with both the different stages of fungal development and the different types of host defense response.