Cytosolic free Ca2+ concentration ([Ca2+]i) was determined in isolated cultured renal LLC-PK1 cells by two independent techniques involving 1) measurement of the fluorescence of an intracellular Ca2+ probe (quin 2) or measurement of the change in extracellular Ca2+ with arsenazo III after making the cell membrane permeable with digitonin, a null-point titration determination. [Ca2+]i determined with the former technique was 98 +/- 5 nM (n = 81) and was 101 +/- 23 nM (n = 7) with the latter. The value of [Ca2+]i was independent of intracellular quin 2 concentration within the range of 0.7-3.0 mM. Increasing extracellular [Ca2+] from 0.5 to 2.0 mM had no effect on [Ca2+]i as determined with quin 2. Ouabain (10(-4) M) or replacement of 120 mM of the extracellular Na+ with choline or lithium resulted in increases in [Ca2+]i. When extracellular [Ca2+] was higher than [Ca2+]i and cell plasma membranes were made permeable with digitonin, Ca2+ was taken up by the intracellular organelles. Ca2+ taken up into the intracellular compartments could be released with carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and Ca2+ uptake could be blocked by ruthenium red, suggesting that the mitochondria were the primary Ca2+-buffering sites under our experimental conditions in the absence of Mg2+ and ATP. The mitochondrial compartment was found to have a very large capacity for Ca2+ buffering. This study represents the first full report using two independent techniques (quin 2 and null-point titration) for determination of [Ca2+]i in epithelial cells and demonstrates excellent agreement between the techniques. The study also demonstrates the utility of these techniques in studying intracellular Ca2+ homeostasis in renal epithelial cells.