The amount of cardiac sarcoplasmic reticulum in rat hearts was estimated by comparing marker activities in the isolated SR fraction with their activities in the homogenate. Four distinguishable markers were measured: the oxalate-supported rate of calcium uptake, the calcium oxalate capacity, 3H-ryanodine binding and the thapsigargin equivalents. The calcium uptake rate and capacity and thapsigargin equivalents were determined in the presence and absence of SR Ca2+ channel blockade with high concentrations of ryanodine. All of these activities are believed to be located only in the SR. However, the calculation of the heart content of SR was somewhat different for the four markers. The calcium uptake rate gave 8.4 mg SR protein per g tissue in the absence of ryanodine, and 9.6 mg per g in its presence; calcium oxalate capacity gave similar numbers, 9.9 mg per g in the absence of ryanodine and 8.0 mg per g in its presence. The thapsigargin titration gave similar equivalent with or without ryanodine, indicating that the homogenate contained about 8.0 mg of SR per g tissue. Using 3H-ranodine binding as a marker, the cardiac content of SR was calculated to be 16.7 mg per g. These differences are attributed to the non-ideal behavior of these markers. Some of the Ca2+ uptake activity is not thapsigargin sensitive, and some of the 3H-ryanodine binding does not fractionate with the SR Ca2+ uptake activity.