The vanilloid receptor TRPV1 is a ligand-gated cation channel that can be activated by capsaicin, acids and noxious heat. For vanilloids, a stretch of approximately 8 amino acids in the vicinity of the TM3 region on the cytosolic side of TRPV1 and R114 and E761 in the N- and C-cytosolic tails, respectively, has been shown to be critical for capsaicin binding and channel activation. Here, we report that intracellular application of vanilloids is insufficient for activating TRPV1 channels in HEK293T cells. Pipette solution (ICS) for recording membrane currents was supplemented with 50 microM capsaicin (n=14) or 1 microM resiniferatoxin (RTX) (n=39) and the responses induced by extracellular capsaicin (1 microM) or RTX (100 nM) were recorded at intervals >50% of that needed for diffusion of Lucifer yellow from the pipette to reach maximum fluorescence (n=7). We found that all cells with expressed TRPV1 exhibited a similar sensitivity to vanilloids irrespective of whether the membrane currents were recorded with electrodes filled with ICS containing capsaicin or RTX or only with control ICS. We suggest that, in addition to intracellularly located agonist recognition sites of TRPV1, there is at least one resides on the extracellular side, which needs to be occupied to activate the channel.