The involvement of vascular fibroblasts (FBs) and smooth muscle (SM)-like cells in physiologic and pathologic processes in large vessels (intimal hyperplasia) and microvessels (capillary arterialization), and the realization that these cells are exposed to interstitial flow shear stress (SS), motivate this study of SS on FB migratory activity. Rat adventitial FBs were grown to either 30-50% confluence (subconfluent FBs; SFBs) or full confluence (confluent FBs; CFBs) in culture. Immunofluorescence and Western blotting assays were conducted to evaluate the expression of two phenotype markers: SM -actin and SM myosin heavy chain (MHC). Both assays indicated a significant increase in SM -actin expression in CFBs compared to SFBs, suggesting a phenotype difference between the two cell populations. SFBs and CFBs both expressed minimal SM MHC. Both cell populations were seeded on Matrigel-coated cell culture inserts and exposed to 4 hours of either 1 or 20 dyn/cm² SS via a rotating disk apparatus in the presence of the chemoattractant PDGF-BB in order to quantify the effect of SS on SFB and CFB migration. Four hours of 20 dyn/cm² SS significantly enhanced SFB migration while it suppressed CFB migratory activity. Four hours of 1 dyn/cm² SS did not significantly alter either SFB or CFB migration levels. Due to the distinct migratory responses of SFBs and CFBs in response to SS, phenotype modulation appears to be one way to regulate their involvement in both physiologic and pathologic remodeling processes.