Transient receptor potential (TRP) channels are important for cellular Ca²⁺ homeostasis and have been described in almost every tissue including skeletal muscle. Immunohistochemical staining has revealed the presence of the cation channels TRPC6 and TRPC3 in the sarcolemma of mouse muscle fibers. The unspecific TRPC channel blocker 2-APB, 100 µM, inhibited background Ca²⁺ influx by more then 50%. Half time of decay of KCl-induced calcium transients was as well significantly influenced by 2-APB. Here we studied if the specific TRPC6 channel blocker ML-9 and/or the TRPC3 blocker Pyr3 affect muscular Ca²⁺ homeostasis. To investigate divalent cation influx we used single interosseus muscle fibers and applied the Mn²⁺ quench technique. Quench of Fura-2 fluorescence was recorded in the presence of 0.5 mM Mn²⁺ (excitation at 360 nm). OAG, an activator of TRPC3/C6 and C7, increased background Ca²⁺ influx nearly twofold (control vs. OAG; 3.3 ±0.3 vs. 6.5±0.6 %/min; n=33; p<0.01), supporting the hypothesis of functional expression of TRPC3 and/or C6 in skeletal muscle fibers. ML-9 application to muscle fibers slowed the quench rate down (control vs. ML-9; 3.6 ±0.4 vs. 2.1±0.3 %/min; n=20; p<0.01), indicating that TRPC6 contributes to background Ca²⁺ influx. The application of the TRPC3 blocker Pyr3 (3 µM) caused as well a marked inhibition of Fura-2 quench rate (control vs. Pyr3; 6.4 ±0.6 vs. 2.5±0.4 %/min; n=46, 46; p<0.01). We conclude that both channels, TRPC3 and TRPC6 are functional in the sarcolemma of mouse muscle fibers. TRPC6 and TRPC3 seem to contribute substantially to background Ca²⁺ influx of muscle fibers at rest and can generate increased divalent cation influx upon activation. The two channels are potential drug targets to treat muscle diseases that are related to disturbed Ca²⁺ homeostasis. This work was supported by the European Commission (FP7-REGPOT-2010, Grant No. 264143) and aktion benni & co e.V.