Objective: Several members of the transient receptor potential (TRP) family of cation channels, among them TRPC6, are expressed in mouse skeletal muscle. Immunohistochemical staining showed a sarcolemmal localization of TRPC6. To test whether TRPC6 is functional in skeletal muscle we applied the specific TRPC6 activator hyperforin and the channel antagonist ML-9 and studied their effects on cytoplasmic Ca²⁺ concentration. Methods: Mouse interosseus muscle fibres were enzymatically dissociated and kept under cell culture conditions for up to 24 h. Cytoplasmic calcium ([Ca²⁺]_i) was monitored in single cells using the Ca²⁺-indicator Fura-2-AM by calculating the ratio of the Fura-2 fluorescence intensity after excitation at 340 and 380 nM. The manganese quench technique was used to estimate the resting calcium influx in muscle fibres. Results: In the presence of extracellular Ca²⁺ the application of 25 mM hyperforin (10 s) caused a long-lasting increase of [Ca²⁺]_i in the fibres. Pre-incubation with ML-9 significantly attenuated the effect of hyperforin (the increase in 340/380 ratio, 260 s after hyperforin application, was 0.032 ± 0.018, n=9 with 100 mM ML-9 vs. 0.076 ± 0.051, n=17 without ML-9; p < 0.01). ML-9 itself had no effect on [Ca²⁺]_i. Without extracellular Ca²⁺ the hyperforin induced rise of [Ca²⁺]_i was reduced to a similar level as observed with ML-9 (increase in 340/380 ratio: 0.041 ± 0.025, n=9). Resting calcium entry was slightly but not significantly decreased in the presence of 100 mM ML-9. Conclusions: The effects of hyperforin and ML-9 indicate that TRPC6 is functionally expressed in mouse muscle fibers. Though endogenous activation mechanisms are unknown, TRPC6 constitutes a potential Ca²⁺ influx pathway in skeletal muscle. However, the channel does not seem to play an important role for the resting Ca²⁺ influx in muscle fibers.