Quantitation of Ca+ and H+ activities within cells using presently available fluorescent probes is optimal when the fluorescence signal is calibrated in situ after each experiment. Fura-2 and 2',7'-bis(2-carboxy-ethyl)-5,6-carboxyfluoroscein (BCECF) are difficult to calibrate in freshly dissociated adult cardiac myocytes because calibration procedures produce cellular hypercontracture. In situ calibration was accomplished in rat ventricular cells by saturating fura-2 with La3+, an agent known to produce myocardial relaxation. Since fura-2 has different spectral properties when complexed with La3+ than with Ca2+, scaling factors were defined in vitro and then verified by experiments in cultured neonatal myocytes. In adult rat myocytes using the La3+ method, intracellular Ca2+ concentration ([Ca2+]i) was 131 +/- 47 nM (n = 14) in quiescent cells; diastolic [Ca2+]i and systolic [Ca2+]i in myocytes stimulated at 1 Hz were 140 +/- 56 and 1,088 +/- 211 nM (n = 5), respectively. BCECF fluorescence was calibrated in situ by a method that prevented cellular hypercontracture and reported a pH value of 7.10 +/- 0.10 in cells stimulated at 1.5 Hz. An additional advantage of both methods is that the buffers employed prevented large changes in the redox state of intracellular pyridine nucleotides, thus preventing a change in cellular autofluorescence during the calibration procedure.