Mammalian counterparts of the Drosophila trp genes are suggested to encode Ca²⁺ permeable cationic channels such as store- and receptor-operated channels (Clapham et al. 2003). TRPC5 is one of the members in the canonical TRP (TRPC) family, which is widely expressed in the cardiovascular (Xu & Beech, 2001), nervous (Greka et al. 2003) and gastrointestinal systems (Lee et al. 2003). Human TRPC5 was cloned in 1999 (Sossey-Alaoui et al. 1999) but its function and pharmacological profile are lacking. 2aminoethoxydiphenyl borate (2-APB) is a widely used agent in the studies of IP3 receptor and store-operated channel signaling. In this study we used patch clamp recordings and Ca²⁺ imaging to examine the effects of 2-APB on human TRPC5 cationic channel stably expressed in HEK 293 cells under a tetracycline-regulated promoter. The experiments were performed at room temperature. Data are expressed as mean ± S.E.M. The unpaired student’s t test was used. The ionic current mediated by TRPC5 was activated by Gd³⁺ at 10 µM. 2-APB inhibited the whole-cell TRPC5 current in a reversible and concentration-dependent manner (IC₅₀ = 19 µM, n = 6). Bath-applied 2-APB (75 µM) also blocked TRPC5 in outside-out (current value at −80 mV was decreased by 67.3 ± 6.9 %, n = 3, P = 0.01) but not inside-out membrane patches (6.2 ± 9.8 %,n = 8, P > 0.05), suggesting an external binding site. Intracellular dialysis of cells with 150 µM 2-APB did not block TRPC5 and extracellular 2-APB remained effective (n = 3). In furaPE3 Ca²⁺ imaging experiments, methoxyverapamil (D600) at 10 µM (n = 60 cells, P > 0.05) or nifedipine at 1 µM (n = 99 cells, P > 0.05) had no effects on TRPC5.The data indicate 2-APB inhibits human TRPC5 channel via the extracellular surface and Ca²⁺ antagonists have no effects on the channel. The presence of extracellular 2-APB binding site, which is distinct from the Ca²⁺ antagonist binding sites of voltage-gated Ca²⁺ channel, may be useful as a new drug target of TRPC5. Clapham DE et al. (2003). Pharmacol Rev 55:591-596. Greka A et al. (2003). Nat Neurosci 6:837-845. Lee YM et al. (2003). Am J Physiol Gastrointest Liver Physiol 284:G604- 616. Sossey-Alaoui K et al. (1999). Genomics 60:330-340. Xu SZ & Beech DJ (2001). J Physiol, 535, 14P-15P. This work was funded by The Wellcome Trust. We are grateful to A.K. Srivastava for human TRPC5 DNA fragments.