Lamellipodium protrusion is linked to actin filament disassembly in migrating fibroblasts [Cramer, 1999: Curr. Biol. 9: 1095–1105]. To further study this relationship, we have identified a method to specifically and sensitively detect G-actin in distinct spatial locations in motile cells using deoxyribonuclease I (DNase I). Although DNase I can bind both G-and F-actin in vitro [Mannherz et al., 1980: Eur. J. Biochem. 95: 377–385], when cells were fixed in formaldehyde and permeabilized in detergent, fluorescently-labelled DNase I specifically stained G-actin and not F-actin. 92–98% of actin molecules were stably retained in cells during fixation and permeabilization. Further, increasing or decreasing cellular G-actin concentration by treating live cells with latrunculin-A or jasplakinolide, respectively, caused a respective increase and decrease in DNase I cell-staining intensity as expected. These changes in DNase I fluorescence intensity accurately reflected increases and decreases in cellular G-actin concentration independently measured in lysates prepared from drug-treated live cells (regression coefficient= 0.98). This shows that DNase I cell-staining is very sensitive using this method. Applying this method, we found that the ratio of G-/F-actin is lower in both the lamellipodium and in a broad band immediately behind the lamellipodium in migrating compared to non-migrating fibroblasts. Thus, we predict that protrusion of the lamellipodium in migrating fibroblasts requires tight coupling to filament disassembly at least in part because G-actin is relatively limited within and behind the lamellipodium. This is the first report to directly demonstrate high sensitivity of cell-staining for any G-actin probe and this, together with the ready commercial accessibility of fluorescently-labelled DNase I, make it a simple, convenient, and sensitive tool for cell-staining of G-actin. Cell Motil. Cytoskeleton 51: 27–38, 2002.© 2002 Wiley-Liss, Inc.