The article introduces Lifeact, a 17-amino-acid peptide that effectively stains filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact does not interfere with actin dynamics in vitro and in vivo, and its chemically modified form allows visualization in nontransfectable cells. The peptide was found to have a low affinity for F-actin, which reduces unwanted perturbations but also limits its use for measuring intra-filament dynamics. Lifeact was successfully tested in various cell types, including mouse embryonic fibroblasts, MDCK cells, primary rat hippocampal neurons, and mouse dendritic cells, showing clear labeling of F-actin. It did not affect cellular processes such as neuronal polarization, lamellipodial flow, or leukocyte chemotaxis. Chemically synthesized F-Lifeact can be used for convenient labeling of fixed samples or nontransfectable cells, providing a cheap and nontoxic alternative to phalloidin. Overall, Lifeact is a versatile marker for visualizing actin dynamics in live and fixed samples, offering a wide range of applications in biomedical research.The article introduces Lifeact, a 17-amino-acid peptide that effectively stains filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact does not interfere with actin dynamics in vitro and in vivo, and its chemically modified form allows visualization in nontransfectable cells. The peptide was found to have a low affinity for F-actin, which reduces unwanted perturbations but also limits its use for measuring intra-filament dynamics. Lifeact was successfully tested in various cell types, including mouse embryonic fibroblasts, MDCK cells, primary rat hippocampal neurons, and mouse dendritic cells, showing clear labeling of F-actin. It did not affect cellular processes such as neuronal polarization, lamellipodial flow, or leukocyte chemotaxis. Chemically synthesized F-Lifeact can be used for convenient labeling of fixed samples or nontransfectable cells, providing a cheap and nontoxic alternative to phalloidin. Overall, Lifeact is a versatile marker for visualizing actin dynamics in live and fixed samples, offering a wide range of applications in biomedical research.