The accurate measurement of ionized intracellular calcium Ca++i in single cells by flow cytometry with the use of a new fluorescent calcium chelator, indo‐1, is described. We have developed a dependable in situ calibration technique that indicates a resting Ca++i in lymphocytes of 100 nM. The enhanced fluorescence of this probe permits its use at sufficiently low cytoplasmic concentrations that buffering of Ca++i transients does not occur. The Ca++i response of small resting B lymphocytes to crosslinking of surface antigen receptors by anti‐immunoglobulin is heterogeneous. With maximal stimulus, the peak Ca++i response occurs in 10 to 20 seconds with most cells reaching levels >/1 μM. Mean Ca++i falls to between 300 and 800 nM by 100 seconds where it remains for more than 10 min. Anti‐δ is a more potent stimulus of increased Ca++i than anti‐μ in terms of both Ca++i level and fraction of B cells responding. Whether this is due to the greater density of surface IgD than IgM, a difference in signal transduction efficiency, or both, is not yet known. Surface immunoglobulin receptors are present in great excess. Less than 3% of surface immunoglobulin is crosslinked at the peak of the Ca++i response.