We used a noninvasive microprobe technique to record in substomatal cavities of barley leaves the apoplastic pH response to different stress situations. When K+ (or Na+) activity at the roots of intact plants was increased from 1 to 50 mM, the leaf apoplastic pH increased by 0.4 to 0.6 units within 8 to 12 min when stomata were open, and within 15 to 20 min when stomata were closed. This reaction was accompanied by a correlative increase in K+ activity. Addition of 1 microM abscisic acid caused an apoplastic alkalinization of 0.5 to 0.8 units, and low temperatures (4 degrees C) increased pH by 0.2 to 0.3 units. Addition of 100 mM sorbitol or pH changes in the range 4.0 to 7.9 had no effect, ruling out that osmotic potential and/or pH is the carried signal. On detached leaves, the same treatments yielded qualitatively similar results, suggesting that the xylem is the most likely signal path. Following the attack of powdery mildew, the apoplastic pH of barley leaves substantially increases. We demonstrate that in susceptible barley, pretreatment (soil drench) with the resistance-inducing chemical benzo- (1,2,3)thiadiazole-7-carbothioic acid S-methyl ester markedly enhances this pH response. This is consistent with previous finding that apoplastic alkalinization is related to the degree of resistance towards this fungus.