Murine epididymal spermatozoa were dispersed in a medium of native osmolality and then transferred to a hypo-osmotic medium to mimic the physiological osmotic challenge, as encountered upon ejaculation into the female tract. The addition of quinine to block sperm K⁺-channels for volume regulation resulted in a size increase of viable cells. Preincubation in 0.1 mM HgCl₂, a standard aquaporin inhibitor, prevented such cell swelling. Addition of the K⁺-ionophore valinomycin to quinine-swollen sperm reversed the swelling, but not after pretreatment of the swollen sperm by HgCl₂. Aqp7, Aqp8, and Aqp9 mRNAs were identified in spermatozoa by RT-PCR, and the entire open reading frames were sequenced and compared with the GenBank database. Western blotting demonstrated specific protein signals for sperm AQP7 and AQP8 expression but probably not AQP9. The role of Hg²⁺-insensitive AQP7, if any, in sperm volume regulation was studied in transgenic mice. Spermatozoa from Aqp7^−/− mice were the same size as wild-type sperm in basal conditions. Quinine-swollen volume, swelling reversal by valinomycin, and inhibition by Hg²⁺ were also similar, indicating efficient water transport in the absence of AQP7. However, both water influx and efflux occurred faster in Aqp7^−/− sperm than wild-type. This faster water movement in the knockout mouse spermatozoa was explainable by an upregulation of Aqp8 expression as revealed by quantitative PCR. Therefore, the Hg²⁺-sensitive AQP8, which was localized in elongated spermatids and spermatozoa, is a likely candidate for a water channel responsible for physiological sperm volume regulation crucial to in vivo fertilization.