Four CatSper ion channel subunit genes (CatSpers 1-4) are required for sperm cell hyperactivation and male fertility. The four proteins assemble (presumably as a tetramer) to form a sperm-specific, alkalinization-activated Ca2+-selective channel. We set out to identify proteins associating with CatSper that might help explain its unique role in spermatozoa. Using a transgenic approach, a CatSper1 complex was purified from mouse testis that contained heat shock protein 70-2, a testis-specific chaperone, and CatSper beta, a novel protein with two putative transmembrane-spanning domains. Like the CatSper ion channel subunits, CatSper beta was restricted to testis and localized to the principal piece of the sperm tail. CatSper beta protein is absent in CatSper1(-/-) sperm, suggesting that it is required for trafficking or formation of a stable channel complex. CatSper beta is the first identified auxiliary protein to the CatSper channel.