The Group VIA Phospholipase A2 (iPLA2β) is the first recognized cytosolic Ca2+-independent PLA2 and has been proposed to participate in arachidonic acid (20:4) incorporation into glycerophosphocholine lipids, cell proliferation, exocytosis, apoptosis, and other processes. To study iPLA2β functions, we disrupted its gene by homologous recombination to generate mice that do not express iPLA 2β. Heterozygous iPLA2β+/- breeding pairs yield a Mendelian 1:2:1 ratio of iPLA2β+/+, iPLA2β+/-, and iPLA2β-/- pups and a 1:1 male:female gender distribution of iPLA2β -/- pups. Several tissues of wild-type mice express iPLA 2β mRNA, immunoreactive protein, and activity, and testes express the highest levels. Testes or other tissues of iPLA2β -/- mice express no iPLA2β mRNA or protein, but iPLA2β-/- testes are not deficient in 20:4-containing glycerophosphocholine lipids, indicating that iPLA2β does not play an obligatory role in formation of such lipids in that tissue. Spermatozoa from iPLA2β-/- mice have reduced motility and impaired ability to fertilize mouse oocytes in vitro and in vivo, and inhibiting iPLA2β with a bromoenol lactone suicide substrate reduces motility of wild-type spermatozoa in a time- and concentration-dependent manner. Mating iPLA2β-/- male mice with iPLA 2β+/+, iPLA2β+/-, or iPLA2β-/- female mice yields only about 7% of the number of pups produced by mating pairs with an iPLA2β +/+ or iPLA2β+/- male, but iPLA 2β-/- female mice have nearly normal fertility. These findings indicate that iPLA2β plays an important functional role in spermatozoa, suggest a target for developing male contraceptive drugs, and complement reports that disruption of the Group IVA PLA2 (cPLA2α) gene impairs female reproductive ability.