High-resolution atomic force microscopy (AFM) was applied to directly observe the dynamic assembly of single actin filaments in HeLa cell extracts in vitro. The F-actin filaments established a dynamic network and formed different types of junctions and branches. The connections of this network were X-, Y- or T-shaped. It was found that the actin filaments were densely covered by endosomes and vesicles from the cell extract, which are thought to stabilize their structures. Using time-lapse AFM, the growth, shrinkage, branching and the interaction of actin filaments with endosomes could be characterized. Our results indicate that the majority of F-actin filaments are static in HeLa extract and that only a minor fraction of filaments undergo dynamic changes. Furthermore, the AFM imaging approach not only provides unique insights into the assembly and dynamics of actin networks; it also builds an avenue to study in vitro assays of complex biological systems.