During stimulation of Dictyostelium discoideum amoebae with the chemoattractant cAMP, extracellular calcium is taken up by the cells. The aim of this study was to determine the cytosolic free calcium concentration ([Ca++]i) during chemotaxis of Dictyostelium cells. In contrast to most vertebrate cells, three major drawbacks were encountered: 1) the indicator fura-2 could not be introduced into the cells by incubation with the ester form, 2) once loaded, the dye was rapidly sequestered into vesicles, 3) the organic anion transport blocker probenecid was not suitable to block sequestration. These problems were met by introducing the indicator into the cells with the scrape-loading technique adapted for use with Dictyostelium and the construction of a new fura-2 derivative, fura-2-dextran. Scrape-loading of Dictyostelium yielded up to 40% of labeled, vital cells. Fura-2-dextran fulfilled the following criteria: 1) it remained homogeneously distributed in the cytoplasm of motile Dictyostelium cells, 2) it retained the fluorescence intensity of fura-2 and the affinity for calcium binding, 3) it was very well suitable to demonstrate changes of [Ca++]i in serum-stimulated fibroblasts. [Ca++]i-measurements with fura-2-dextran in chemotactically active D. discoideum amoebae revealed that the large decrease in the extracellular calcium concentration is not accompanied by an overall change in [Ca++]i. Chemotaxis in this organism occurs in the absence of global changes in [Ca++]i. However, we cannot exclude either short-lived or local changes just beneath the plasma membrane.