31P NMR was used to study the dynamics of phosphate pools during substrate utilization by aerobic and anaerobic suspensions of the yeast Candida utilis and by aerobic suspensions of the yeast Brettanomyces intermedius. In both yeast, the cytoplasmic pH was monitored; in C. utilis also the vacuolar pH could be measured. When glucose was used as a substrate for C. utilis, the vacuolar store of inorganic phosphorus (both orthophosphate and polyphosphate) was mobilized to replenish cytoplasmic phosphate which had become very low due to the build-up of high sugar phosphate levels. The hydrolysis of polyphosphate was glucose-dependent; it did not occur with ethanol as the substrate. After glucose depletion resynthesis of polyphosphate occurred only under aerobic conditions; anaerobic C. utilis cells continued to hydrolyze vacuolar polyphosphate. This difference between the aerobic and anaerobic suspension could be related to differences in cellular ATP levels. When ethanol was employed as a substrate, both Candida utilis and Brettanomyces intermedius exhibited a substantial increase in polyphosphate levels. These observations suggested a dual role for polyphosphate in yeasts both as a phosphate and an energy store. The cytoplasmic pH in C. utilis displayed characteristic responses to metabolic changes during glucose degradation. B. intermedius experienced a strong cytoplasmic acidification upon ethanol utilization due to acetic acid formation. The mechanism of transport of Pi across the vacuolar membrane in C. utilis appeared to be different from that reported for the plasma membrane.