1. To characterize the effect of clofibric acid (Clof) on the Ca(2+) release mechanism in the sarcoplasmic reticulum (SR) of skeletal muscle, we analysed the properties of Clof-induced Ca(2+) release under various conditions using chemically skinned skeletal muscle fibres of the mouse. 2. Clof (>0.5 mM) released Ca(2+) from the SR under Ca(2+)-free conditions buffered with 10 mM EGTA (pCa >8). 3. Co-application of ryanodine and Clof at pCa >8 but not ryanodine alone reduced the Ca(2+) uptake capacity of the SR. Thus, Ca(2+) release induced by Clof at pCa >8 must be a result of the activation of the ryanodine receptor (RyR). 4. At pCa >8, (i) Clof-induced Ca(2+) release was inhibited by adenosine monophosphate (AMP), (ii) the inhibitory effect of Mg(2+) on the Clof-induced Ca(2+) release was saturated at about 1 mM, and (iii) Clof-induced Ca(2+) release was not inhibited by procaine (10 mM). These results indicate that Clof may activate the RyR-Ca(2+) release channels in a manner different from Ca(2+)-induced Ca(2+) release (CICR). 5. In addition to this unique mode of opening, Clof also enhanced the CICR mode of opening of RyR-Ca(2+) release channels. 6. Apart from CICR, a high concentration of Ca(2+) might also enhance the unique mode of opening by Clof. 7. These results suggest that some features of Ca(2+) release activated by Clof are similar to those of physiological Ca(2+) release (PCR) in living muscle cells and raise the possibility that Clof may be useful in elucidating the mechanism of PCR in skeletal muscle.