ABSTRACT The flightless I protein contains an actin-binding domain with homology to the gelsolin family and is likely to be involved in actin cytoskeletal rearrangements. It has been suggested that this protein is involved in linking the cytoskeletal network with signal transduction pathways. We have developed antibodies directed toward the leucine rich repeat and gelsolin-like domains of the human and mouse homologues of flightless I that specifically recognize expressed and endogenous forms of the protein. We have also constructed a flightless I-enhanced green fluorescent fusion vector and used this to examine the localization of the expressed protein in Swiss 3T3 fibroblasts. The flightless I protein localizes predominantly to the nucleus and translocates to the cytoplasm following serum stimulation. In cells stimulated to migrate, the flightless I protein colocalizes with β-tubulin- and actin-based structures. Members of the small GTPase family, also implicated in cytoskeletal control, were found to colocalize with flightless I in migrating Swiss 3T3 fibroblasts. LY294002, a specific inhibitor of PI 3-kinase, inhibits the translocation of flightless I to actin-based structures. Our results suggest that PI 3-kinase and the small GTPases, Ras, RhoA and Cdc42 may be part of a common functional pathway involved in Fliih-mediated cytoskeletal regulation. Functionally, we suggest that flightless I may act to prepare actin filaments or provide factors required for cytoskeletal rearrangements necessary for cell migration and/or adhesion.