Measurement of intracellular ionized calcium concentrations ([Ca2+]i) has been indispensable in elucidating the central role of [Ca2+]i as a trigger of cellular responses to activating stimuli. Such studies have employed the dye quin2, which has not been readily adapted to analysis of individual small cells. We show here that the calcium response of large numbers of single cells can be analyzed with the use of flow cytometry and the recently described dye, indo-1. Such analyses demonstrate for the first time the heterogeneous nature of the [Ca2+]i response to mitogenic stimuli within populations of peripheral blood lymphocytes (PBL). By simultaneous quantitation of one- or two-color surface immunofluorescence labels, some of this heterogeneity of [Ca2+]i response in PBL is shown to be related to cellular immunophenotype. Almost all T cells responded to anti-CD3 antibody; however, the response is greater among CD4+ than CD8+ cells, and within the CD4+ population the rate of response to stimulation by antibody to CD3 differed between subpopulations defined by expression of the common leukocyte marker p220. In contrast, not all T cells responded to phytohemagglutinin (PHA), even at very high doses. As with anti-CD3, after stimulation with PHA, CD4+ cells showed a larger proportion of responding cells than did CD8+ cells. In separate experiments, indo-1 was found not to impair reproductive viability of PBL, thereby providing the potential for analysis of functional activity after the separation of cells by sorting on the basis of the [Ca2+]i response to stimuli. Mixing experiments indicated that a response of a subpopulation representing as little as 0.3% of total cells could be readily detected. Thus, the flow cytometric assay with indo-1 is the first technique that allows the quantitative analysis of response differences of small subpopulations of cells and intercellular variation in [Ca2+]i.