1. Cardiac sarcoplasmic reticulum (SR) was prepared by differential centrifugation from euthyroid, hyperthyroid, hypothyroid as well as (131)I-treated plus thyroxine-substituted rabbits. The function of the isolated SR has been characterized by measuring the ATP-dependent calcium uptake, the calcium storing capacity, the calcium concentrating ability and the calcium-dependent ATP hydrolysis by the calcium-activated ATPase in the presence of oxalate.2. The rate of calcium uptake and the rate of the calcium-dependent ATP hydrolysis (calcium-activated ATPase) by the SR were significantly increased in hyperthyroidism, whilst both activities were markedly reduced in hypothyroidism. Thyroxine administration to (131)I-treated animals prevented a decrease in the rate of calcium uptake as well as in the rate of the calcium-dependent ATP hydrolysis by the calcium-activated ATPase.3. The transport ratio (rate of calcium uptake divided by the rate of calcium-dependent ATP hydrolysis) of SR preparations from euthyroid controls was 0.93, suggesting a stoicheiometry of calcium uptake and calcium-activated ATP split of 1.0. The transport ratio was unchanged in one hyper- and hypothyroid group, whilst a small but significant decrease or increase was observed after an excessive thyroxine treatment of a prolonged state of hypothyroidism, respectively.4. The saturation kinetics of calcium transport by the SR were described by Michaelis-Menten kinetics. The maximum rate of calcium uptake (V) was 0.193+/-0.004, 0.223+/-0.002 and 0.124+/-0.003 mumole Ca(2+)/mg protein. min (means +/-S.E.) for euthyroid, hyperthyroid and hypothyroid SR, respectively. The Michaelis constants (K(m)) were (2.87+/-0.30) x 10(-7)M (2.68+/-0.15) x 10(-7)M and (4.00+/-0.48) x 10(-7)M for the euthyroid, hyperthyroid and hypothyroid SR, respectively (means +/-S.E.). The K(m) values for the hyper- and hypothyroid SR were not significantly different from controls.5. The calcium storing capacity as well as the calcium concentrating ability of the SR was unaltered at different levels of thyroid activity.6. The steady-state level of calcium was the same for SR isolated from euthyroid, hyperthyroid and hypothyroid rabbits, indicating that calcium influx and calcium efflux are strongly coupled at steady-state filling of the SR.7. It is suggested that the increased or reduced rate of calcium transport by the SR in hyperthyroidism or hypothyroidism, respectively, found in vitro, might be at least partially responsible for the shortening of the relaxation time of cardiac muscle in the hyperthyroid state and the prolongation of the relaxation time in the hypothyroid state observed in vitro and in vivo.