Type 2 Toll-like receptors (TLR2s) are expressed in cell membranes and recognize a wide range of pathogen-associated molecular patterns derived from bacteria, such as lipoteichoic acid (LTA). The aim of this study was to evaluate the effect of TLR2 activation by LTA on the activity of type 1 Na(+)/H(+) exchanger (NHE) in T84 intestinal epithelial cells. Short-term (0.5 hour) and long-term (18 hours) TLR2 activation significantly inhibited NHE1 activity in a concentration-dependent manner (0.01-100 µg/ml; -7 ± 3 to -21 ± 3% and 3 ± 3 to -21 ± 3% of control values, respectively). S3226 [3-[2-(3-guanidino-2-methyl-3-oxopropenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride], an NHE3-selective inhibitor, did not affect the inhibitory effect on NHE activity. LTA-induced NHE inhibition did not occur in the presence ofethylisopropylamiloride (an NHE1 inhibitor). Long-term TLR2 activation decreased NHE1 affinity for Na(+) (Km= 64.98 ± 1.67 mM) compared with control (Km= 20.44 ± 0.54 mM) without changes in Vmax values. After TLR2 activation, we observed tyrosine-protein kinase (SRC) activation, phosphatidylinositol 3-kinase (PI3K) recruitment, and adenylyl cyclase (AC3) phosphorylation. The total amount of AC3 increased (23 ± 8% of control) after long-term treatment with LTA. Anti-AC3 small interfering RNA prevented LTA-induced NHE1 inhibition, similar to that observed with the AC3 inhibitor KH7 [(±)-2-(1H-benzimidazol-2-ylthio)propanoic acid 2-[(5-bromo-2-hydroxyphenyl)methylene]hydrazide]. A significant increase in cAMP levels (32 ± 3% and 14 ± 2% after short- and long-term stimulation, respectively) was detected, and inhibition of protein kinase A (PKA), phospholipase C (PLC), and downregulation of protein kinase C (PKC) prevented NHE1 inhibition. Inhibition of nuclear factor-κΒ (NF-κB) failed to revert NHE1 inhibition. We concluded that activation of TLR2 reduces NHE1 activity in epithelial cells through an alternative pathway that is unrelated to NF-κB, which involves SCR, PI3K, AC3, PKA, PLC, and PKC.