The effects of NaCl stress on cell area and intracellular pH (pH(i)) of individual cells of two Debaryomyces hansenii strains were investigated. Our results show that one of the strains was more NaCl tolerant than the other, as determined by the rate of growth initiation. Whereas NaCl stress caused similar cell shrinkages (30-35%), it caused different pH(i) changes of the two D. hansenii strains; i.e., in the more NaCl-tolerant strain, pH(i) homeostasis was maintained, whereas in the less NaCl-tolerant strain, intracellular acidification occurred. Thus, cell shrinkage could not explain the different intracellular acidifications in the two strains. Instead, we introduce the concept of yeasts having an intracellular pK(a) (pK(a,i)) value, since permeabilized D. hansenii cells had a very high buffer capacity at a certain pH. Our results demonstrate that the more NaCl-tolerant strain was better able to maintain its pK(a,i) close to its pH(i) homeostasis level during NaCl stress. In turn, these findings indicate that the closer a D. hansenii strain can keep its pK(a,i) to its pH(i) homeostasis level, the better it may manage NaCl stress. Furthermore, our results suggest that the NaCl-induced effects on pH(i) were mainly due to hyperosmotic stress and not ionic stress.