The properties of a negative transient signal (negative peak) observed during the first seconds of the induction of the photoacoustic (PA) signal in dark-adapted barley leaves treated with methyl viologen (MV) and diuron and then exposed to high temperatures have been examined. Under those conditions no electron donation from photosystem II (PSII) occurred, and electron flow through PSI could be supported only by soluble reductants located in the chloroplast stroma. The negative peak was observed only if the PA signal had been monitored at low, and not high, frequencies. The peak obviously originated from the oxygen consumption by PSI. The size of the peak increased as the temperature of preheating was raised from 39 to 45°C. The size of the peak decreased exponentially with a half-time of 3.7 s during illumination under low light. This decrease was found to be much faster under strong light. The recovery of the peak during dark acclimation required several minutes. It is concluded that the negative peak reflects the oxygen consumption supported by stromal reductants, their pool being rapidly exhausted under light in the presence of MV. The maximal size of the pool was calculated as 140 eq:P700 in dark-adapted leaves.