The accuracy of flow cytometric measurement of intracellular calcium with fluo‐3 is compromised by variation in basal fluorescence intensity due to heterogeneity in dye uptake or compartmentalization. We have loaded cells simultaneously with fluo‐3 and SNARF‐1. When SNARF‐1 fluorescence is collected at approximately 600 nm, its intensity does notchange upon cell activation. Furthermore, fluo‐3 and SNARF‐1 fluorescence signals exhibit a linear relationship. The ratio of fluo‐3 to SNARF‐1 eliminates a significant proportion of variation in fluorescence intensity caused by variation in fluo‐3 uptake and thus can be used as a sensitive parameter for measuring changes in [Ca2+]i. Copyright © 1990 Wiley‐Liss, Inc.