Calcium flux measurements of different subpopulations of cells by flow cytometry are important in understanding complex interactions in the immune system. This paper discusses the use of the difference of Log signals as a preferred method for obtaining this information simultaneously with other immunofluorescence parameters. We describe simple modifications to a commercial instrument that enables the measurement of calcium flux in addition to three immunofluorescence paramein. Finally, we show an application of this technique to measuring calcium flux of T cell subsets in human blood. We show that different subsets of peripheral CD4 T cells have significantly different capabilities to flux calcium after CD3 stimulation. These differences are related to the functional capacities of the cells within these subsets. © 1995 Wiley-Liss, Inc.