The effects of three-dimensional (3-D) culture and cyclic stretch stimulation on the expression of contractile proteins were investigated in freshly isolated rat aortic smooth muscle cells (FSMC). Primary cells were cultured statically on cell culture dishes (two-dimensional (2-D) culture) or in type I collagen gel matrix (3-D culture). Changes in their expression level of actin filaments (AFs) and smooth muscle myosin heavy chain (SM-MHC) were measured quantitatively using an accurately-calibrated fluorescent microscopy. The expression of AFs and SM-MHC decreased in both cultures in their early stages. Cell morphology was quite different between the two cultures: the cells had a flattened and irregular shape in the 2-D culture, while they had a fusiform shape with a well-defined long axis in the 3-D. Nineteen-day culture in the gel significantly increased the expression levels of AFs and SM-MHC while the expression levels remained low in the 2-D. Further and early increase in the expression levels was observed in the cells cultured in the gel with cyclic stretch of ∼8% amplitude and 1 Hz frequency. The cyclic stretch also induced alignment of FSMCs in the gel parallel to the stretch direction, and the cell alignment was observed earlier than the increase in their contractile proteins. These results indicate that the 3-D culture in collagen gel may increase the expression level of contractile proteins in FSMCs while maintaining their fusiform morphology, and cyclic stretch may efficiently increase the expression levels when the cells aligned in the stretch direction.