Inflammation accompanies muscle injury. Protease-Activated Receptors (PARs) are a novel class of G-protein coupled receptors activated by serine proteases that play a prominent role in the inflammatory response. PAR expression has been demonstrated in skeletal myotubes, and PARs predominantly signal via the phospholipase C (PLC) pathway, which is also found in skeletal myotubes. In this study, we examined the effect of PAR stimulation on electrically evoked Ca2+ transients elicited in skeletal C2C12 myotubes. C2C12 myoblasts were grown on collagen-coated cover slips and incubated in Dulbeccos modified Eagles medium with 20% foetal calf serum at 37°C and 5% CO2. Cytosolic Ca2+ was measured with fura-2 using a Cairn spectrophotometer connected to an inverted epifluorescence microscope. The myotubes were stimulated electrically via platinum electrodes connected to a pulse stimulator (0.3 ms pulse duration at 0.1 Hz). The serine protease thrombin caused a brief transient increase in the resting [Ca2+], and a sustained decrease in the peak of electrically evoked SR Ca2+ transients in C2C12 myotubes. Ca2+ transient peaks dropped to 51.4 ± 9.8% (n=15) of initial values after thrombin exposure, compared to a drop to 97.1 ± 1.7% of initial values with no thrombin exposure (n=15, p<0.001). The effect on the Ca2+ transients continued for up to 60 minutes. Synthetic peptides that specifically activate PAR receptors without proteolytic cleavage revealed PAR-1 as the receptor involved. The effect of thrombin on the Ca2+ transients was prevented by the PLC blocker U37122, implicating involvement of the PLC pathway in the thrombin-mediated effect. These results show that thrombin can decrease sarcoplasmic reticulum Ca2+ release in skeletal muscle cells, and could play a role in the muscle weakness observed during injury-induced inflammation.