Saccharomyces cerevisiae cells contain two intracellular and soluble trehalases with distinct subcellular location (cytosol and vacuoles, respectively). Both enzymes showed an opposite pattern of activity along the growth cycle. Activity of the cytosolic trehalase was high in cells growing exponentially on fermentable sugars (glucose, mannose or galactose) and sharply decayed as the cultures enter stationary phase coinciding with the beginning of trehalose biosynthesis. By contrast, vacuolar trehalase was only detectable in glucose-grown resting cells or in cultures growing on respiratory substrates (glycerol or ethanol). This enzyme was partially derepressed in the mutant hex2, which is deficient in glucose repression. Addition of fresh YPD medium to stationary-phase cultures induced the sudden reactivation of cytosolic trehalase with the concomitant slower inactivation of vacuolar trehalase. However, addition of glucose or various nitrogen sources alone had only a minor effect on both activities. The presence of cycloheximide had no effect on cytosolic trehalase, whereas completely blocked the appearance of vacuolar trehalase suggesting the requirement of protein synthesis 'de novo'.