Like many yeasts, bacteria, and other sporulating microorganisms, Acanthamoeba castellanii (Neff), a free-living amoeba with pathogenic relatives, differentiates into a dormant form when deprived of nutrients. Acanthamoeba cysts redifferentiate into trophozoites when food is resupplied. We report here that Acanthamoeba encystment is also triggered by elevated osmolarity, and that osmolarity and cell surface receptor binding are synergistic in triggering differentiation. Additions of sodium chloride or glucose to rich growth media were used to produce specific osmolarity increases and similar encystment results were obtained with either additive. Although many organisms, including Acanthamoeba and mammalian cells, have been shown to adapt to hyperosmolar conditions, this is the first demonstration that hyperosmolarity can be a primary differentiation signal.