Abstract: The aim of this study was to establish a method of isolating and culturing smooth muscle cells from the ductus deferens of rats. Smooth muscle cells were prepared from ductus deferens by explanting technique after dissection of adventitia and intimae, and cultured in vitro. The identification of the smooth muscle cells were verified by using anti α-smooth muscle actin (α-SMA) immunohistochemistry studies. The result suggested that the cells are multi-morphous, showing long fusiform or star shapes. The apophysis of cells contacted and coalesced to each other, in some regions the cells overlapped in multilayer, while in the other regions they formed monolayer that fluctuated and showed a “peak-valley” shape. They presented a positive reaction through immunohistochemistry studies. The purity of the cells was more than 99% through this method. The culturing of smooth muscle cells by explanting technique is simple and stable.