We have found using imaging techniques that stimulating Jurkat human leukaemic T-cells with ionomycin in the presence of FM1-43, a dye used to monitor exocytosis and endocytosis, causes large (6-10-fold) increases in FM1-43 fluorescence. These responses are too large to be caused by exocytosis. Instead, three lines of evidence suggest that FM1-43 is responding to phospholipid scrambling. First, ionomycin also stimulates increases in the fluorescence of annexin V, a phosphatidylserine-specific probe, while thapsigargin does not stimulate fluorescence increases of either probe. Secondly, cells that exhibit FM1-43 fluorescence increases after ionomycin stimulation stain with annexin V once FM1-43 is washed out. Thirdly, ionomycin stimulates uptake of 7-nitrobenz-2-oxa-1,3-diazole-labelled phosphatidylcholine, a specific assay for scramblase activity, whereas thapsigargin does not. We find that FM1-43 reports phospholipid scrambling with ‘better’ kinetics than annexin V, and does require extracellular Ca²⁺ to report phospholipid scrambling. We suggest that FM1-43 may be a useful probe to study the dynamics of phospholipid scrambling. The results are the first demonstration that FM1-43 can respond significantly to a biological process other than vesicular trafficking.