We have recently demonstrated the involvement of phospholipase D (PLD) in actin polymerization during mammalian sperm capacitation. In the present study, we investigated the involvement of phosphatidylinositol 3- and 4-kinases (PI3K and PI4K) in actin polymerization, as well as the production of PIP_2(4,5), which is a known cofactor for PLD activation, during bovine sperm capacitation. PIK3R1 (p85 α regulatory subunit of PI3K) and PIKCB (PI4K β) in bovine sperm were detected by Western blotting and immunocytochemistry. Wortmannin (WT) inhibited PI3K and PI4K type III at concentrations of 10 nM and 10 μM, respectively. PI4K activity and PIP_2(4,5) production were blocked by 10 μM WT but not by 10 nM WT, whereas PI3K activity and PIP_3(3,4,5) production were blocked by 10 nM WT. Moreover, spermine, which is a known PI4K activator and a component of semen, activated sperm PI4K, resulting in increased cellular PIP_2(4,5) and F-actin formation. The increases in PIP_2(4,5) and F-actin intracellular levels during sperm capacitation were mediated by PI4K but not by PI3K activity. Activation of protein kinase A (PKA) by dibutyryl cAMP enhanced PIP_2(4,5), PIP_3(3,4,5), and F-actin formation, and these effects were mediated through PI3K. On the other hand, activation of PKC by phorbol myristate acetate enhanced PIP_2(4,5) and F-actin formation mediated by PI4K activity, while the PI3K activity and intracellular PIP_3(3,4,5) levels were reduced. These results suggest that two alternative pathways lead to PI4K activation: indirect activation by PKA, which is mediated by PI3K; and activation by PKC, which is independent of PI3K activity. Our results also suggest that spermine, which is present in the ejaculate, regulates PI4K activity during the capacitation process in vivo.