Sperm motility is regulated by a complex balance between kinases and phosphatases. Among them, phosphatidylinositol 3-kinase (PI 3-kinase) has been recently suggested to negatively regulate sperm motility (Luconi, M., Marra, F., Gandini, L., Lenzi, A., Filimberti, E., Forti, G. and Baldi, E. (2001). Hum. Reprod. 16, 1931-1937). We demonstrate the presence and activity of PI 3-kinase in human spermatozoa and have investigated the molecular mechanism(s) by which the PI 3-kinase inhibitor, LY294002, triggers an increase in sperm motility. PI 3-kinase inhibition results in an increase in intracellular cAMP levels and in tyrosine phosphorylation of the protein kinase A-anchoring protein AKAP3. These effects finally result in a stimulation of protein kinase A (PKA) binding to AKAP3 in sperm tails through the regulatory subunit RIIbeta. The increased binding of RIIbeta to AKAP3 induced by LY294002 is mainly due to tyrosine phosphorylation of AKAP3, since it is completely blocked by the tyrosine kinase inhibitor erbstatin, which also reverses the effects of LY294002 on motility and suppresses PKA-AKAP3 interaction. The requirement of PKA binding to AKAP3 for sperm motility is confirmed by the reduction of motility induced by an inhibitor of RIIbeta-AKAP3 binding, Ht31, whose effects on sperm motility and PKA binding to AKAP3 are reversed by LY294002. These results demonstrate that PI 3-kinase negatively regulates sperm motility by interfering with AKAP3-PKA binding, providing the first evidence of a molecular mechanism by which PKA can be targeted to sperm tails by interaction with tyrosine phosphorylated form of AKAP3.