Fluorescent Ca(2+) indicators have been extremely valuable in understanding the role of intracellular Ca(2+). However, the presence of extracellular dye can confound interpretation of data due to indicator accumulation in the Ca(2+)-rich medium, which induces an increase in the fluorescence signal. By using a mathematical approach, we show that overlooking extracellular dye usually leads to overestimating cytosolic Ca(2+) ([Ca(2+)]) levels. We propose an experimental design and provide mathematical formulations to make the appropriate correction. We applied our model to determine [Ca(2+)] in Fluo-3-loaded bovine aortic endothelial cells (BAECs). Our results indicate that for basal level Ca(2+), the uncorrected value overestimates by a factor of 2.7 the result obtained when extracellular dye was accounted for. We also showed that both bradykinin (BK) and ATP significantly increase [Ca(2+)] in BAECs. For the uncorrected values, BK and ATP induced 2.3- and 3.3-fold apparent increases in [Ca(2+)], respectively. When applying the correction, there was a 4.5- and 5.4-fold induction of [Ca(2+)] for BK and ATP, respectively. Our theoretical and experimental models provide explanations and, at least in part, solutions to the dye leakage problem, and should thus be a valuable tool in clarifying the proper usage of fluorescent dyes for Ca(2+) measurements.