ABSTRACT. The germination of microsporidian spores under conditions expected to affect water flow across the plasma membrane-wall complex was studied by assessing their responses to in vitro stimulation with Na+ or K+. Partial or full substitution of common water with D2O, which more effectively coats ions and electrostatically-charged cell surfaces with relatively stable hydration layers, delayed and inhibited spore germination in a concentration-dependent manner; yet, preincubation in 100% D2O did not change the normal response to standard stimulation. Water structure-breaking conditions, such as an increase in temperature (within the 15° C to 40° C range) or in ionic strength (2- to 10-fold normal), opposed the inhibition by D2O and allowed significant stimulation by Li+, the monovalent cation with the largest hydration diameter and a usually weak stimulant action on the spores. Ethanol, known to reduce water permeation across cell membranes and phospholipid bilayers, also caused a powerful and dose-dependent (1% to 4% v/v) inhibition of spore germination, but pretreatment with ethanol did not affect the normal response. HgCl2, an inhibitor of specific water channels, blocked spore germination at just 250 μM in the normal stimulation solution irrespective of the temperature, and permitted only a delayed response in high salt stimulation solutions. However, the inhibition by Hg2+ was abolished by the simultaneous presence of 2-mercaptoethanol in the medium. These results suggest (1) that spore germination is keenly dependent upon the hydration states of both the plasma membrane-wall complex and the stimulant ions, and (2) that osmotic water flows into the spores through specific transmembrane pathways with critical sulfhydryl groups, i.e. analogous to the water channels that facilitate water movements across the plasma membranes of highly permeable cells.