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Formin-homology-domain-containing proteins interact with Rho-family GTPases and regulate actin cytoskeleton organization and gene transcription. FHOD1 is a member of this family, interacts with Rac1 and induces transcription from the serum response element. In this study, we examined the effects of FHOD1 expression on cytoskeletal organization and function in mammalian cells. FHOD1 proteins were stably expressed in WM35 melanoma cells and NIH-3T3 fibroblasts. Cells expressing full-length FHOD1 demonstrated an elongated phenotype compared with vector-transfected cells and cells expressing a truncated FHOD1 (1-421) that lacks the conserved FH1 and FH2 domains. Full-length FHOD1 co-localized with filamentous actin at cell peripheries. Cells transiently expressing a C-terminal FHOD1 truncation mutant (ΔC, residues 1-1010), which lacks an autoinhibitory protein-protein interaction domain, displayed prominent stress fibers. FHOD1 (1-421) did not induce stress fibers but localized to membrane ruffles in a manner similar to the full-length protein, indicating that the FH1 and FH2 domains are required for stress fiber appearance. FHOD1 ΔC (1-1010)-dependent stress fibers were sensitive to dominant-negative RacN17 and the RhoA and ROCK inhibitors, C3 transferase and Y-27632. Stable overexpression of full-length FHOD1 enhanced the migration of WM35 and NIH-3T3 cells to type-I collagen and fibronectin, respectively. Cells expressing FHOD1 (1- 421) migrated similar to control cells. Integrin expression and activation were not affected by FHOD1 expression. Moreover, FHOD1 overexpression did not alter integrin usage during adhesion or migration. These data demonstrate that FHOD1 interacts with and regulates the structure of the cytoskeleton and stimulates cell migration in an integrin-independent manner.