A number of previous studies have suggested the involvement of phosphoinositide 3-kinase (PI3K) in Toll-like receptor (TLR) signaling. However, there have also been a number of conflicting reports. The PI3K inhibitor wortmannin greatly enhanced TLR-mediated inducible nitric-oxide synthase (iNOS) expression and cytokine production in the mouse macrophage cell line Raw264.7. The effect of wortmannin was common to TLR2, -3, -4, and -9 and was accompanied by activation of nuclear factor-kappaB and up-regulation of cytokine mRNA production. We were surprised to find that another PI3K inhibitor, LY294002, strongly suppressed the production of iNOS and cytokines. This effect of 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) was based on its inhibitory effect on mRNA synthesis. Expression of dominant-negative mutants of PI3K in macrophages augmented the lipopolysaccharideinduced expression of iNOS. Introduction of a pH1 vector producing short hairpin RNA that targets a catalytic subunit of PI3K (p110beta) also enhanced the TLR-mediated responses. Thus, the augmentation of TLR signals by wortmannin was mediated through the inhibition of PI3K, whereas the effect of LY294002 was not explained by its effect on PI3K. These discrepancies in the effects of pharmacological inhibitors in TLR-signaling may have caused confusion regarding the role of PI3K in innate immunity.