Interleukin (IL)-6 production in contracting skeletal muscle and IL-6 concentration in plasma are increased after prolonged and strenuous exercise. However, as tissue stress or damage are unspecific triggers of increased cytokine levels, we examined whether moderate muscle activity is an independent stimulus for cytokine expression, and to which extent invasive procedures might affect the results. Soleus muscles were isolated from sedentary rats or from rats that had been running on a treadmill at moderate intensity (70% of maximal oxygen uptake) for 1 h. In another group the soleus muscle was prepared in situ and stimulated intermittently at 5 Hz for 1 h, so that maximal developed force declined by 30%. In situ prepared soleus muscles not subjected to electrical stimulation were used as controls. Messenger RNA (mRNA) expression of 11 cytokines was analysed in the soleus muscles using multiprobe RNAse protection assay, and IL-6 plasma concentration was measured by enzyme-linked immunosorbent assay. Treadmill exercise did not affect the mRNA expression of any of the measured cytokines in the soleus muscle. Irrespective of electrical stimulation, mRNA expression of IL-6 and IL-1beta were significantly increased in the surgically manipulated soleus muscles. Interleukin-6 plasma concentration was not affected by treadmill running or electrical stimulation. Conclusion, gentle surgical manipulation is a strong stimulus for IL-6 and IL-1beta mRNA synthesis in skeletal muscle, whereas exercise or electrical muscle stimulation at moderate intensity does not independently affect cytokine mRNA levels in the contracting soleus.