We have studied the role of phosphatidylinositol 3-kinases (PI 3-kinases) in the regulation of the actin cytoskeleton in MTLn3 rat adenocarcinoma cells. Stimulation of MTLn3 cells with epidermal growth factor (EGF) induced a rapid increase in actin polymerization, with production of lamellipodia within 3 min. EGF-stimulated lamellipodia were blocked by 100 nM wortmannin, suggesting the involvement of a class Ia PI 3-kinase. MTLn3 cells contain equal amounts of p110␣ and p110, and do not contain p110␦. Injection of specific inhibitory antibodies to p110␣ induced cell rounding and blocked EGF-stimulated lamellipod extension, whereas control or anti-p110 antibodies had no effect. In contrast, both antibodies inhibited EGF-stimulated DNA synthesis. An in situ assay for actin nucleation showed that EGF-stimulated formation of new barbed ends was blocked by injection of anti-p110␣ antibodies. In summary, the p110␣ isoform of PI 3-kinase is specifically required for EGF-stimulated actin nucleation during lamellipod extension in breast cancer cells.PI 3-kinases 1 are important signaling intermediates in a variety of regulated cellular processes (1). They are classified based on their regulation and substrate specificity (2). Class I enzymes produce PI[3]P, PI[3,4]P 2 , and PI[3,4,5]P 3 , whereas class II and III enzymes produce PI[3]P and PI[3,4]P 2 , or only PI[3]P, respectively. Class Ia enzymes exhibit the greatest diversity of the known PI 3-kinases, with multiple isoforms of both the regulatory (p85) and catalytic (p110) subunits (2). Differential phosphorylation of p85␣ and p85 and differential activation of p85␣-and p85-associated PI 3-kinase have been reported (3, 4). Knockouts of p85␣ further suggest that the p85␣ and p85 are not redundant (5, 6). Distinct class Ia catalytic subunit isoforms also have different functions. Both p110␣ and p110 play a role in mitogenesis, although p110␣ is required for responses to a broader range of growth factors (7,8). Recently, distinct signaling properties for p110 isoforms have been demonstrated in macrophages (9).We examined the specific functions of p110␣ and p110 in MTLn3 cells, a metastatic variant of the 13762NF rat mammary adenocarcinoma. MTLn3 cells undergo chemotaxis in an EGF gradient (10,11). This response involves the actin-dependent extension of a lamellipod in the direction of increasing EGF concentrations, with a zone of newly polymerized F-actin at the leading edge (12). Using isoform-specific inhibitory antibodies against p110␣ and p110, we now show that EGFstimulated lamellipod extension requires p110␣ but not p110. Significantly, anti-p110␣ antibodies blocked the formation of new barbed ends during an in situ actin nucleation assay. These studies provide direct evidence that p110␣ is required for the regulation of actin nucleation by EGF.