AbstractThe Arp2/3 complex‐mediated assembly and protrusion of a branched actin network at the leading edge occurs during cell migration, although some studies suggest it is not essential. In order to test the role of Arp2/3 complex in leading edge protrusion, Swiss 3T3 fibroblasts and Jurkat T cells were depleted of Arp2 and evaluated for defects in cell morphology and spreading efficiency. Arp2‐depleted fibroblasts exhibit severe defects in formation of sheet‐like protrusions at early time points of cell spreading, with sheet‐like protrusions limited to regions along the length of linear protrusions. However, Arp2‐depleted cells are able to spread fully after extended times. Similarly, Arp2‐depleted Jurkat T lymphocytes exhibit defects in spreading on anti‐CD3. Interphase Jurkats in suspension are covered with large ruffle structures, whereas mitotic Jurkats are covered by finger‐like linear protrusions. Arp2‐depleted Jurkats exhibit defects in ruffle assembly but not in assembly of mitotic linear protrusions. Similarly, Arp2‐depletion has no effect on the highly dynamic linear protrusion of another suspended lymphocyte line. We conclude that Arp2/3 complex plays a significant role in assembly of sheet‐like protrusions, especially during early stages of cell spreading, but is not required for assembly of a variety of linear actin‐based protrusions. Cell Motil. Cytoskeleton 2008. © 2008 Wiley‐Liss, Inc.