Cellular alignment studies have shown that cell orientation has a large effect on the expression and behavior of cells. Cyclic strain and substrate microtopography have each been shown to regulate cellular alignment. This study examined the combined effects of these two stimuli on the alignment of bovine vascular smooth muscle cells (VSMCs). Cells were cultured on substrates with microgrooves of varying widths oriented either parallel or perpendicular to the direction of an applied cyclic tensile strain. We found that microgrooves oriented parallel to the direction of the applied strain limited the orientation response of VSMCs to the mechanical stimulus, while grooves perpendicular to the applied strain enhanced cellular alignment. Further, the extent to which parallel grooves limited cell alignment was found to be dependent on the groove width. It was found that for both a small (15microm) and a large (70microm) groove width, cells were better able to reorient in response to the applied strain than for an intermediate groove width (40microm). This study indicates that microtopographical cues modulate the orientation response of VSMCs to cyclic strain. The results suggest that there is a range of microgroove dimensions that is most effective at maintaining the orientation of the cells in the presence of an opposing stimulus induced by cyclic strain.