Following entry into the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that results in competence to fertilize ova. Associated with capacitation is an increase in membrane conductance to both Ca 2+ and K + , leading to an elevation in cytosolic Ca 2+ critical for activation of hyperactivated swimming motility. In mice, the Ca 2+ conductance (alkalization-activated Ca 2+ -permeable sperm channel, CATSPER) arises from an ensemble of CATSPER subunits, whereas the K + conductance (sperm pH-regulated K + current, KSPER) arises from a poreforming ion channel subunit encoded by the slo3 gene (SLO3) subunit. In the mouse, both CATSPER and KSPER are activated by cytosolic alkalization and a concerted activation of CATSPER and KSPER is likely a common facet of capacitation-associated increases in Ca 2+ and K + conductance among various mammalian species. The properties of heterologously expressed mouse SLO3 channels differ from native mouse KSPER current. Recently, a potential KSPER auxiliary subunit, leucine-rich-repeat-containing protein 52 (LRRC52), was identified in mouse sperm and shown to shift gating of SLO3 to be more equivalent to native KSPER. Here, we show that genetic KO of LRRC52 results in mice with severely impaired fertility. Activation of KSPER current in sperm lacking LRRC52 requires more positive voltages and higher pH than for WT KSPER. These results establish a critical role of LRRC52 in KSPER channels and demonstrate that loss of a non-pore-forming auxiliary subunit results in severe fertility impairment. Furthermore, through analysis of several genotypes that influence KSPER current properties we show that in vitro fertilization competence correlates with the net KSPER conductance available for activation under physiological conditions. sperm fertility | KSPER | SLO3 channels | auxiliary subunits U pon entry into the female reproductive tract, mammalian sperm undergo a sequence of maturational steps, collectively termed capacitation, to become competent to fertilize an egg (1, 2). Two important components of this process, thought to be shared among mammalian species, are cytosolic alkalization (3-5) and then an associated increase in cytosolic Ca 2+ (6, 7). These events are coupled with changes in ionic fluxes in sperm membrane. Over the past 10 y, the application of patch-clamp recording to individual sperm (8) has allowed identification of ionic currents that respond to alkalization and/or Ca 2+ (9-12). In mouse sperm, alkalization leads to activation of two spermspecific channels, the Ca 2+ -permeable CATSPER channel (8-10, 13) and the K + -permeable KSPER K + channel (14, 15). KSPER and CATSPER currents are also present in human sperm (16-18), although intriguingly there seem to be differences in regulation of each channel type between mice and humans (11,12,17).Despite the species-specific differences in the details of their regulation, KSPER and CATSPER are of central importance in sperm function and fertility in both humans and mice. In mouse spe...