Endothelial cells (ECs) in athero-protective regions are elongated with actin and microtubule fibers aligned parallel to the direction of blood flow. Fluid shear stress (FSS) affects EC shape and functions, but little is known about shape-dependent EC properties that are independent of FSS. To evaluate these properties, ECs were elongated on micropatterned (MP) 25 mu m wide collagen-coated lanes (MPECs) and characterized by cell shape index, actin and microtubule alignment, and polarization of the microtubule-organizing center (MTOC). ECs on non-patterned surfaces were also exposed to FSS. MPEC elongation was microtubule-dependent (and actin-independent); shape indices and cytoskeletal alignment were comparable to FSS-elongated ECs. Cytoskeletal alignment was lost when MPECs were exposed to perpendicular FSS, but not parallel FSS. MTOC polarization was FSS-dependent. Thus, by isolating EC elongation and cytoskeletal alignment from FSS, micropatterning creates a platform for studying EC shape-related cellular functions that are independent of FSS. (c) 2008 Elsevier Inc. All rights reserved.