This communication reports the development of a modified procedure for chemical loading of aequorin in small multicellular cardiac preparations, with special emphasis directed toward the implementation of a new method for computer-controlled low-photon counting and digital processing and analysis of the data to obtain intracellular Ca2+ concentration ([Ca2+](i)). In eight ferret right ventricular trabeculae, we measured the mechanical performance and found that, at 1.25 mM extracellular Ca2+ concentration ([Ca2+](o)), resting tension, developed tension, and time to peak tension were unchanged by the loading procedure. Estimated resting and peak systolic [Ca2+](i) were 299 ± 65 and 766 ± 131 nM, respectively. Thirty minutes after raising the [Ca2+](o) to 5 mM, there was a robust increase in mechanical performance, with peak systolic [Ca2+](i) averaging 1,218 ± 222 nM. The diastolic [Ca2+](i) remained unchanged. In four other trabeculae, exposure to a low-Na+-containing superfusate demonstrated a remarkable beat- to-beat correspondence of increases in diastolic [Ca2+](i) and resting tensions. The same beat-to-beat concordance was also observed between the rapidly changing amplitudes of peak [Ca2+](i) and developed tension. In additional experiments, simultaneous recordings of [Ca2+](i) and force transients were obtained during rapid pace pause maneuvers. These studies showed distinct and quantifiable fluctuations of [Ca2+](i) in a 1:1 relation to the mechanical record to a frequency of at ~300 beats/min. These results demonstrate that beat-to-beat measurements of [Ca2+](i) and tension transients can be obtained with good resolution in multicellular cardiac preparations.