We measured cytoplasmic free calcium in protoplasts of Citrus sinensis Osb. Protoplasts were isolated from cell-suspension cultures grown as microcalli. The cell surface presented acid polysaccharide filaments, probably responsible for cell adhesion. These filaments were disrupted by enzymatic digestion liberating the cells and subsequently the protoplasts. Protoplasts were loaded with the pentapotassium salt of indo-1 by incubation in acidic solutions of this calcium indicator. Loading increased with decreasing pH and osmotica. Dye loaded in this manner was distributed only in the cytoplasm. The protoplast autofluorescence was very low and did not interfere with measurements. Cytoplasmic free-calcium concentration in Citrus protoplasts, using microspectrofluorimetry, was estimated to be 154 nmol.L-1. This resting value was increased when protoplasts were exposed to high extracellular Ca2+ (1 mmol.L-1) or 6BA (1 mu mol.L-1) suggesting that the pathway in cytokinin signal transduction in these cells may involve a Ca2+ uptake facilitation from extracellular medium.1513329333