Constitutive exocytosis was investigated in the yeast Saccharomyces cerevisiae using temperature-sensitive mutant (sec) strains which do not allow vesicle fusion to the plasma membrane at the restrictive temperature. Secretory vesicles were accumulated in the cell at the restrictive temperature and then protein synthesis was blocked with cycloheximide. Upon returning the cells to the permissive temperature the contents of the accumulated vesicles were secreted. This allowed the study of constitutive exocytosis independent of the processes responsible for vesicular biosynthesis. Neither the kinetics nor magnitude of exocytosis were affected by removal of external Ca2+ or perturbations of cytosolic Ca2+. This suggests that in those systems where calcium is required for exocytosis it is a regulatory molecule and not part of the mechanism of membrane fusion. Release occurred over a very broad range of pH and in media with different ionic compositions, suggesting that ionic and potential gradients across the plasma membrane play no role in exocytosis in yeast. High osmolarity inhibited the rate, but not the extent, of release. A novel inhibitory effect of azide was detected which occurred only at low pH. Vanadate also inhibited release in a pH-independent manner. Secretion occurred at the same rate in cells with or without accumulated vesicles. This infers a rate-limiting step following vesicle accumulation, perhaps a limiting number of release sites on the plasma membrane.