The studies presented here characterize a simple, quantitative NBDphallacidin extraction assay for determining the F-actin content of fMLP-activated neutrophils. The NBDphallacidin extraction assay is based upon the specificity of NBDphallacidin binding to F-actin and the solubility of NBDphallacidin in methanol. Cells are fixed, permeabilized, and stained with NBDphallacidin; the cells are then pelleted, the bound NBDphallacidin is extracted into methanol, and the RFI (excite 465; emit 535) of the solution is determined. Binding of NBDphallacidin to neutrophils is saturable and 90% of bound NBDphallacidin is displaced by nonfluorescent phalloidin. The extraction of bound NBDphallacidin into methanol is complete and the excitation/emission characteristics of NBDphallacidin are not altered by extraction. The assay is relatively inexpensive, applicable to the study of cells in suspension or on substratum, allows kinetic studies with 5-10s time resolution, and is not affected by the shape of the cell or the distribution of the probe. We used the NBDphallacidin extraction assay to study the kinetics of fMLP-induced change in the F-actin content of neutrophils and the effect of tBOC peptide, an inhibitor of fMLP binding, on these changes. The extraction assay reveals a rapid, sequential fMLP-induced increase followed by a decrease in F-actin content. The tBOC peptide inhibits fMLP-induced actin polymerization. Addition of tBOC during fMLP-induced polymerization or at times when F-actin content is maximal enhances F-actin depolymerization. The rate of F-actin depolymerization is greater than or equal to fourfold faster in the presence than in the absence of tBOC. The results show that The NBDphallacidin extraction assay is useful for studying the kinetics of change in F-actin content of nonmuscle cells; fMLP receptor occupancy is required for fMLP-dependent polymerization but not depolymerization; and both the actin polymerizing and depolymerizing processes are active in the cell within 5 s after fMLP stimulation. Implications of these observations for understanding the observed increase and, then, decrease in F-actin content of fMLP-activated cells are discussed.