Intracellular free calcium concentration ([Ca2+]i) was measured in Langendorff-perfused ferret hearts (30 degrees C, pH 7.4) by loading paced hearts with the 19F NMR calcium indicator, the 5,5'-difluoro derivative of 1,2-bis(o-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid (5FBAPTA), to an initial cytosolic concentration of approximately 120 microM. Increasing the pacing frequency raised the end-diastolic [Ca2+]i from 299 +/- 44 nM (mean +/- SEM) at 0.2 Hz to 522 +/- 54 nM at 1.0 Hz and 691 +/- 166 nM at 2.0 Hz. Raising [Ca]o from 1.8 to 7.0 mM at a pacing frequency of 1.0 Hz increased end-diastolic [Ca2+]i to 625 +/- 39 nM. In unpaced hearts perfused with diltiazem (100 microM), [Ca2+]i fell rapidly to a steady-state value of < 100 nM after 60 min. Raising [Ca]o from 1.8 to 7.0 mM had no detectable effect on resting [Ca2+]i. The time course of the [Ca2+]i transient was measured in hearts paced at 1.1 Hz and perfused with 1.8 mM [Ca]o. The peak [Ca2+]i was approximately 2 microM at approximately 150 msec after the pacing pulse, and peak developed LVP occurred at 550 msec compared with 280 msec in control hearts not loaded with 5FBAPTA. Comparisons with data obtained by other techniques, including fluorescent [Ca2+]i indicators, imply that although the end-diastolic [Ca2+]i values obtained with 5FBAPTA in beating hearts are elevated by the concentrations of intracellular 5FBAPTA required for signal detection, the changes in [Ca2+]i observed in response to experimental interventions are qualitatively consistent with previous data.