1. The effect of diltiazem on isolated sarcoplasmic reticulum (SR) from rabbit skeletal muscle was studied. To observe calcium movement into and out of the SR, a fluorescent chelate probe technique with chlortetracycline (CTC) as a reagent was employed. 2. Tris-ATP-induced calcium accumulation by the isolated SR was associated with a rise in the CTC fluorescence. The effect of ATP was dose dependent. 3. Diltiazem (6 x 10(-4)M, 2 x 10(-3)M) prevented ATP-induced calcium accumulation by the SR. 4. Addition of EGTA to the media chelates external calcium and caused calcium release that can be reversed by further addition of calcium chloride. Similarly diltiazem caused a rapid release of accumulated calcium from the SR, which is not reversed by the addition of calcium chloride. 5. It seems that the effect of diltiazem may be related to SR membrane-bound calcium being available for release.