Prolonged exercise decreased the rate of Ca+ release in sarcoplasmic reticulum (SR) vesicles isolated from rat muscle by 20–30% when release was initiated by 5, 10, and 20 μM AgNO3. [3H]Ryanodine binding was also depressed by 20% in SR vesicles isolated from the exercised animals. In contrast, the maximum amount of Ca2+ released by Ag+ remained unaffected by exercise. The passive permeability of SR vesicles and the rate of Ca2+ release in the presence of ruthenium red, a known inhibitor of the Ca2+ release mechanism, was not affected by prolonged exercise. These results suggest that exercise depressed Ca2+ release from SR by directly modifying the Ca2+ release channel.