TRPC5 is a member of the Transient Receptor Potential protein family and is widely expressed, including in vascular smooth muscle cells (Xu et al., 2006). It has calcium channel properties and is activated by different stimuli, showing polymodal behaviour (Zeng et al., 2004). In this study our aim was to investigate the mechanism linking G-protein coupled receptors to TRPC5 activation. Our hypothesis is that the coupling involves generation of LPC via the activation of Ca²⁺-independent PLA₂ (iPLA₂), which catalyze the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate a free fatty acid (e.g. arachidonic acid) and 2-lysophospholipid. LPC is suggested as a convergence point for some stimulatory signals (Wolf et al., 1997) and we recently showed extracellular and intracellular activation by LPC, but not arachidonic acid (Flemming et al., 2006). Receptor agonist activating TRPC5 include carbachol (CCh) and sphingosine-1-phosphate (S1P) (Xu et al., 2006, Zeng et al. 2004). We now find that priming TRPC5 channels with LPC abolishes the stimulatory effect of CCh consistent with LPC being involved in the receptor-activation mechanism. To further investigate this effect we firstly treated cells with 25µM of bromoenol lactone (BEL), an irreversible inhibitor of iPLA₂. Significant reduction in TRPC5 activation by CCh or S1P was observed in BEL-treated cells. However LPC activation of TRPC5 was not affected. Secondly we used an iPLA₂ specific siRNA transfection method to suppress iPLA₂ gene expression, confirming specific knock down by real time RT-PCR. Again CCh and S1P activation of TRPC5 were inhibited, whereas LPC activation was not. The data support the idea that receptor-activation of TRPC5 occurs via iPLA₂ and the generation of LPC, which then acts relatively directly on TRPC5.