Threshold behavior is an important aspect of signal transduction pathways that allows for responses to be turned on or off. Human neutrophil responses to N-formyl peptides, including oxidant production and release, exhibit threshold behavior with respect to the number of G proteins available for signaling; progressive treatment of neutrophils with pertussis toxin causes the conversion of responding cells to nonresponding cells. To quantify the threshold level of G proteins required for signaling of N-formyl peptide stimulated oxidant production in a neutrophil population, we used a plasma membrane associated G protein quantification assay in conjunction with a sorting flow cytometer and measured differences in the average number of G proteins available for signaling per cell in both the responding and the nonresponding subpopulations after pertussis toxin treatment. Although there appeared to be a threshold separating responding cells and nonresponding cells for a given sample, no discrete threshold was measured across multiple treatment conditions. A mathematical model of the early steps in signaling suggests that cell-to-cell variability in signal parameters, such as numbers of signal components and values of kinetic rate constants, obscures the measurement of a discrete threshold and leads to an apparent decrease in the threshold level of G proteins available for signaling as the total G proteins are decreased.