We have studied the effects of acute administration of tri-iodothyronine (T3) on cytosolic free calcium levels [Ca2+]i in single rat myocytes microinjected with aequorin. Ventricular myocytes were isolated by perfusing rat hearts with collagenase, and healthy, rod-shaped cells were injected to <1% of their volume with aequorin. The photons emitted from single cells were measured and a conversion to [Ca2+]i made on the basis of an in vitro calibration after the remaining aequorin had been discharged by cell lysis. Only cells that depolarized reversibly (showing elevated [Ca2+]i levels) when superfused with 80mM KC1, and which gave a substantial signal on lysis with distilled water were used. The [Ca2+]i rose from a resting value of 150±56nM (mean ± SD, n=14) by 127±47nM on depolarization with 80mM KC1. Application of T3 (1-100nM) led to an increase (P<0.05) in [Ca2+]i (mean amplitude of 152±35nM) before returning to baseline. The median duration of these events was 10 min (range = 1.4-34.4 min). The time to response was shorter when lOOnM T3 was applied (median and range; 6.8, 0-14 min) than when 1nM T3 was used (16, 7.0-56.1 min) (P<0.05). To conclude, physiological concentrations of thyroid hormones caused rapid but transient stimulation of [Ca2+]i in single rat myocytes.