Vasoactive intestinal peptide (VIP) was originally isolated as a vasodilator intestinal peptide, then as a neuropeptide. In the immune system, VIP is described as an endogenous macrophage-deactivating factor. VIP exerts its immunological actions in a paracrine and/or autocrine manner, through specific receptors. However, very little is known about the molecular regulation of VIP type 2 receptor (VPAC(2)) in the immune system. We now report that different toll-like receptor (TLR) ligands selectively regulate the VPAC(2) receptor gene and show a gene repression system controlled by key protein kinase signalling cascades in macrophages. VPAC(2) gene expression is regulated by gram-positive (TLR2 ligands) and gram-negative bacteria wall constituents (TLR4 ligands). Moreover, VPAC(2) is tightly regulated TLR2- or TLR2/6- but not TLR2/1-mediated mechanisms are responsible for the induction of VPAC(2). TLR stimulation by viral or bacterial nucleic acids did not modify the VPAC(2) mRNA levels. Remarkably, imiquimod--a synthetic TLR7 ligand--led to a potent up-regulation of VPAC(2) gene expression. TLR5 stimulation by flagellin present in gram-positive and gram-negative bacteria did not affect VPAC(2) mRNA. The p38 mitogen-activated protein kinase (MAPK) activity accounted for the TLR4-mediated induction of VPAC(2) gene expression. Surprisingly, our data strongly suggest for the first time a tightly repressed control of VPAC(2) mRNA induction by elements downstream of MAPK kinase 1/2, PI3K/Akt, and particularly Jun-NH(2)-terminal kinase signalling pathways.