A pma1-1 mutant of Saccharomyces cerevisiae with reduced H(+)-ATPase activity and the isogenic wild-type strain accumulated high levels of trehalose in response to a temperature upshift to 40 degrees C and after addition of 10% ethanol, but only modest levels in response to a rapid drop in external pH and after addition of decanoic acid. There was, however, no correlation between the absolute levels of trehalose in the stressed cells and their viability. All these treatments induced a significant decrease in intracellular pH, and surprisingly, this decrease was very similar in both strains, indicating that intracellular acidification could not be the triggering mechanism for trehalose accumulation in response to stress. A careful investigation of metabolic parameters was carried out to explain how trehalose accumulated under the four different stress conditions tested. No single and common mechanism for trehalose accumulation could be put forward and the transcriptional activation of TPS1 was not unequivocally related to trehalose accumulation. Another finding was that a pma1-1 mutant exhibited a two- to threefold greater capacity to accumulate trehalose than the isogenic wild-type. This enhanced disaccharide synthesis could be attributed to a twofold higher trehalose-6-phosphate synthase activity, together with a fourfold higher content of intracellular UDP-Glc. In addition, this mutant showed 1.5-fold higher levels of ATP compared to the wild-type. The various stress treatments studied showed that a drop in intracellular pH does not correlate with trehalose accumulation. It is suggested that plasma membrane alteration could be the physiological trigger inducing trehalose accumulation in yeast.