Assessment of the regulation of plant metabolism by the calcium ion requires a knowledge of its intracellular levels and dynamics. Technical problems have prevented direct measurement of the concentration of intracellular Ca2+ in plant cells in all but a few cases. In this study we show that electropermeabilized protoplasts of Daucus carota and Hordeum vulgare took up the Ca2+ indicating fluorescent dye methoxyquinoline(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (Quin-2) and the Ca2+ indicating photoprotein, aequorin. These protoplasts subsequently recovered their plasma membrane integrity. However, up to 10% of intracellularly trapped Quin-2 was associated with a protoplast vacuolar fraction. Also, Quin-2 loading reduced total ATP levels by approximately 60% and inhibited subsequent protoplast division whereas aequorin loading reduced ATP content by only 20% and did not prevent division. Therefore, the basal cytoplasmic Ca2+ level measured with aequorin (less than 200 nanomolar) may more reliably reflect that found in vivo in the unperturbed protoplast than that measured with Quin-2 (120-360 nanomolar). However, measurements made with aequorin were found to be inaccurate at Ca2+ levels below 200 nanomolar, Quin-2 proving complementary in indicating these low Ca2+ concentrations. Cytosolic Ca2+ was observed to increase on treatment with azide and silver ions.