Fluorescent Ca indicators have been extremely valuable in understanding intracellular [Ca] ([Ca]i) regulation in many cell types. The calibration of these indicators in the intracellular environment, however, has been a continuous challenge. We performed in vivo calibrations of indo-1 in isolated rabbit ventricular myocytes loaded with the acetoxymethylester form of indo-1 and used the perforated patch variation of whole cell voltage clamp. Voltage, [Na], and [K] gradients were eliminated to approach equilibrium. We also took advantage of the powerful Na/Ca exchange in cardiac myocytes so that [Ca]i would be equilibrated with [Ca]o (because there was no [Na] or voltage gradient). The equilibration of [Na] and [Ca] across the membrane was tested by measuring the reversal potential of Na current and poking the cell to test for changes in [Ca]i-dependent fluorescence ratio. The apparent dissociation constant, Kd for indo-1 in the cellular environment was 844 nM, which is approximately 2–3 times higher than that in aqueous solutions. In a separate series of experiments, a null point approach was used to determine the [Ca]i in intact cells at rest for very long periods (82 +/- 6 nM). This is lower than that measured 15 s after a train of steady-state twitches ([Ca]i = 294 +/- 53 nM). These experiments also allowed the direct assessment of the shortening versus [Ca]i relationship in intact cells.