Single ventricular myocytes were isolated by collagenase digestion from the hearts of 6-8-month-old male Wistar rats in either the control (euthyroid) state or after 7 days of daily injection of 0.64 mg/kg thyroxine (hyperthyroid). Myocytes were field-stimulated from slack length, and contraction was measured with an inverted microscope-photodiode array-computer apparatus. The effect of pacing rate and ouabain administration on systolic and diastolic function was examined. Single myocytes isolated from hyperthyroid hearts maintain the properties of bulk muscle, because maximal twitch velocity is augmented 98% and the time course of contraction as measured by the time to peak shortening, relaxation time, or contraction duration is abbreviated 39%. Spontaneous sarcoplasmic reticulum calcium release, as measured by the occurrence of contractile waves, is increased in the hyperthyroid myocytes. This increased frequency of spontaneous sarcoplasmic reticulum calcium release is most marked under conditions known to be associated with high intracellular calcium, such as low pacing rates or digitalis glycoside administration. It can account for the hypoperformance of the hyperthyroid myocytes noted under these conditions because it is associated with depletion of sarcoplasmic reticulum calcium stores and diminution of subsequent twitch amplitude. These observations may help explain, in part, the cellular basis of the altered cardiac performance in the hyperthyroid state.