Trehalase activity in a yeast protoplast lysate increased approximately 40-times upon preincubation with cAMP and ATP. The activity present without the preincubation could all be sedimented at 8000 x g, for 10 min confirming the previously reported localization of the active trehalase (Ta) in the vacuoles. Virtually all the trehalase activity newly formed upon the preincubation, however, was found in the soluble fraction, indicating that a trehalase-zymogen (Tz) is located in the cytosol. This raises the possibility that a cAMP-dependent phosphorylation not only transforms Tz to Ta but also initiates the transfer of trehalase from the cytosol into the vacuoles.