A method for visualization of cytosolic Ca super(2+) distribution was applied to living plant tissue. A mixture of the fluorescent probes Fluo-3 and Fura Red was used. The emitted fluorescence was scanned simultaneously in two channels with a laser-scanning confocal microscope and rationing was performed. The homogeneity of the Fluo-3/Fura Red concentration ratio throughout the tissue after AM-ester loading was proven. In vitro calibration permitted conversion of Fluo-3/Fura Red fluorescence ratios to Ca super(2+) values. Apparent K sub(D) of 286 nM, R sub(min) of 0.43 and R sub(max) of 18 were calculated. The in vivo determination of extreme ratio values was performed by permeabilizing the plasmalemma for Ca super(2+) with a ionophore and manipulating the extracellular Ca super(2+). The resultant R sub(minv) of 1.33 and R sub(maxv) of 2.69 for vegetative apices, and R sub(mini) of 1.26 and R sub(maxi) of 3.45 for apices induced to flowering, suggested incomplete equalization of extra- and intracellular Ca super(2+) levels in these experiments. In Chenopodium rubrum, the cytosolic Ca super(2+) patterns of apical tissue obtained using Fluo-3 and Fura Red were significantly different between vegetative apices and apices after photoperiodic flower induction. This methodological approach may also be helpful for studying cytosolic Ca super(2+) distribution in other living plant tissues.