Transient receptor potential (TRP) channels play a fundamental role in signal transduction in response to hormones, temperature, osmolarity and intracellular messengers. Today, the lack of specific activators and blockers for individual TRP channels makes it difficult to study their role in native cells. TRPC6, a member of the canonical TRPC subfamily can be activated by G-protein coupled receptors linked to phospholipase C. Further, TRPC6 can be directly activated by diacylglycerol, its derivate OAG (oleoyl-2-acetyl-sn-glycerol) and independently by protein kinase C induced phosphorylation. Electrophysiological data indicated that the myosin light chain inhibitor ML-9 (1-(5-chloronaphthalene-1-sulphonyl) homopiperazine, HCl) is a candidate for a TRPC6 channel blocker. To test this hypothesis we investigated the effect of ML-9 on OAG induced and carbachol (CCh) induced Ca²⁺ transients in HEK 293 cells expressing TRPC6. Transient rises of cytoplasmic Ca²⁺ ([Ca²⁺]_i) were monitored in single cells using the Ca²⁺-indicator Fura-2-AM by calculating the ratio of the Fura-2 fluorescence intensity after alternating excitation at 340 and 380 nM. In our cell model the application of OAG (100 mM) typically caused rises of [Ca²⁺]_i that developed within several seconds and declined to baseline within 80 seconds. OAG had no effect on non-transfected HEK 293 cells. In the presence of 100 mM ML-9 the OAG-induced calcium transients were nearly completely suppressed, i.e. their amplitudes were less than 5% of control. ML-9 application alone did not affect [Ca²⁺]_i in TRPC6 expressing cells. Not only OAG but also CCh (100 mM) could be used to elicit Ca²⁺ transients in TRPC6 expressing HEK 293 cells. In contrast to OAG, CCh also induced Ca²⁺ transients in non-transfected HEK cells, indicating a mechanism of [Ca²⁺]_i increase that does not necessarily require TRPC6 activity. In the presence of ML-9 the CCh-induced calcium transients were unaffected, both in TRPC6 expressing cells and in non-transfected HEK 293 cells. These results support the hypothesis that ML-9 is a TRPC6 channel blocker. Since ML-9 has no direct stimulatory effect on [Ca²⁺]_i and does not interfere with carbachol induced changes of [Ca²⁺]_i it seems to be a rather specific TRPC6 blocker in our cell model.