Based on the enhancement of rhodamine phalloidin fluorescence after its binding to actin filaments we have developed a technique to quantify F-actin, drastically (> 100 times) reducing consumption of the expensive fluorescent dye and sample material in comparison to previous methods. Depolymerization of F-actin is prevented by utilizing short incubation times and stabilization of the filaments by actin-binding proteins or formaldehyde. Equilibrium and kinetic mathematical models relating rhodamine fluorescence with F-actin concentrations were used to predict the optimal assay conditions. The method has been applied to measure relative and absolute F-actin concentrations in cytosolic fractions and stimulus-induced actin polymerization in neutrophils. The cells were lysed with octy1-beta-D-glucopyranoside, which is compatible with the assay due to its high critical micelle concentration. As the assay takes less than 1 h and eliminates all previously required washing or extraction steps, it is faster and much simpler than any other presented up to now for quantification of filamentous actin. Moreover, the method is unique for reliable and easy F-actin measurements in cell-free systems.