Aquaporins mediate rapid selective water transport across biological membranes. Elucidation of their precise physiological roles promises important insight into cellular and organismal osmoregulation. The genome of the yeast Saccharomyces cerevisiae encodes two similar but differentially regulated aquaporins. Here, we show that expression of AQY1 is stimulated during sporulation and that the Aqy1 protein is detectable exclusively in spore membranes. When spores are rapidly frozen, those that lack Aqy1 survive better, providing for a functional test of active spore water channels. Under ambient conditions, lack of Aqy1 reduces spore fitness. Because this reduction is independent from germination conditions, Aqy1 may be important during spore formation rather than subsequent maintenance or germination. Indeed, it seems that Aqy1 is degraded after spores have been formed and during germination. Taken together, Aqy1 is developmentally controlled and may play a role in spore maturation, probably by allowing water outflow. Taken together, we demonstrate a functional role of an aquaporin in gametogenesis, as well as in the formation of durable structures such as spores, a role that may have wider biological and medical implications.