Lifeact is a 17-amino-acid peptide that efficiently labels filamentous actin (F-actin) in eukaryotic cells and tissues without interfering with actin dynamics. It can be used in non-transfected cells and is chemically modified to include a fluorophore, allowing for visualization of actin dynamics. Lifeact-GFP, a fusion with green fluorescent protein, was tested in various cell types, including mammalian cells, and showed effective labeling of F-actin. It was found to have a low affinity for F-actin, which allows it to be used as a non-toxic alternative to phalloidin for actin labeling. Lifeact-GFP did not affect cytoskeletal functions such as retrograde flow in fibroblasts, chemotaxis of dendritic cells, or neuronal polarization. It was also effective in fixed samples and live cells, showing good overlap with phalloidin staining. Lifeact is a small, cost-effective probe that can be synthesized quickly and is suitable for a wide range of applications in actin imaging. It is a versatile marker for visualizing F-actin in various biological contexts.Lifeact is a 17-amino-acid peptide that efficiently labels filamentous actin (F-actin) in eukaryotic cells and tissues without interfering with actin dynamics. It can be used in non-transfected cells and is chemically modified to include a fluorophore, allowing for visualization of actin dynamics. Lifeact-GFP, a fusion with green fluorescent protein, was tested in various cell types, including mammalian cells, and showed effective labeling of F-actin. It was found to have a low affinity for F-actin, which allows it to be used as a non-toxic alternative to phalloidin for actin labeling. Lifeact-GFP did not affect cytoskeletal functions such as retrograde flow in fibroblasts, chemotaxis of dendritic cells, or neuronal polarization. It was also effective in fixed samples and live cells, showing good overlap with phalloidin staining. Lifeact is a small, cost-effective probe that can be synthesized quickly and is suitable for a wide range of applications in actin imaging. It is a versatile marker for visualizing F-actin in various biological contexts.