Fat1, an atypical cadherin induced robustly after arterial injury, has significant effects on mammalian vascular smooth muscle cell (VSMC) growth and migration. The related Drosophila protein Fat interacts genetically and physically with Atrophin, a protein essential for development and control of cell polarity. We hypothesized that interactions between Fat1 and mammalian Atrophin (Atr) proteins might contribute to Fat1 effects on VSMCs. Like Fat1, mammalian Atr expression increased after arterial injury and in VSMCs stimulated with growth and chemotactic factors including angiotensin II, basic fibroblast growth factor, and platelet-derived growth factor BB. Two distinct Atr2 transcripts, atr2L and atr2S, were identified by Northern analysis; in VSMCs, atr2S mRNA expression was more responsive to stimuli. By immunocytochemistry, Fat1 and Atrs colocalized at cell-cell junctions, in the perinuclear area, and in the nucleus. In coimmunoprecipitation studies, Fat1 interacted with both Atr1 and Atr2; these interactions required Fat1 amino acids 4300-4400 and an intact Atro-box in the Atrs. Knock-down of Atrs by small interfering RNA did not affect VSMC growth but had complex effects on migration, which was impaired by Atr1 knockdown, enhanced by Atr2L knockdown, and unchanged when both Atr2S and Atr2L were depleted. Enhanced migration caused by Atr2L knockdown required Fat1 expression. Similarly, orientation of cells after monolayer denudation was impaired in cells with Atr1 knockdown but enhanced in cells selectively depleted of Atr2L. Together these findings suggest that Fat1 and Atrs act in concert after vascular injury but show further that distinct Atr isoforms have disparate effects on VSMC directional migration.