Calcium uptake was measured in homogenates and microsomal preparations enriched in sarcoplasmic reticulum vesicles isolated from hearts of hypertrophic (BIO 14.6) and dilated (BIO 53.58) cardiomyopathic as well as control (F1B) Syrian hamsters at 3, 7, 9, and 11 months of age. Calcium uptake studies were done using the Millipore filtration technique under conditions known to restrict transport to the sarcoplasmic reticulum. Steady-state calcium uptake capacity was used as a measure of the relative amounts of sarcoplasmic reticulum in homogenates prepared from individual hearts. At 3 months of age, there were no differences in calcium uptake in homogenates from control or myopathic hearts. However, by 9 months, although calcium uptake of homogenates from control and hypertrophic hearts was the same, calcium uptake by homogenates from dilated hearts was significantly depressed both in initial rate and capacity. Similar trends were seen in the microsomal vesicle preparations, but the decrease in calcium uptake in the dilated hearts was significantly lower by 3 months of age. The catalytic activity of the sarcoplasmic reticulum transport enzyme was estimated from the ratio of velocity to capacity, which provides a measure of the fractional rate of filling of the sarcoplasmic reticulum with calcium. The velocity-to-capacity ratios were not different at any of the ages in both the homogenate and microsomal preparations. The results of this study demonstrate that a major defect in the dilated cardiomyopathy may be due to a decrease in the volume or number of sarcoplasmic reticulum calcium transport sites rather than a decrease in specific activity of the transport enzyme.