Stem cells have a key role in regenerative medicine and tissue engineering. Although not immortal, they are able to expand manyfold in culture retaining at the same time their growth and multilineage potential. They also show a migratory capacity when transplanted systemically in animal model with injuries. Thanks to their properties and their plasticity stem cells are of great importance since they can be used as a tool for repair damaged tissues and organs. Mesenchymal stem cells, in particular, have the ability to differentiate into lineages of mesodermal tissues, such as skeletal muscle, bone, tendons, cartilage, and fat under appropriate culturing conditions. Recent evidence suggest that the adipose tissue is a promising source of mesenchymal stem cells attracting the interest of researchers and clinicians. It is rich of pluripotent stromal cells, available in large amounts and more readily accessible than bone marrow. Furthermore, comparative analysis of mesenchymal stem cells of bone marrow and adipose tissue show that cells are not different regarding morphology, immune phenotype, success rate of isolating and differentiation ability. Our experience at the Experimental Zooprophylactic Institute of Sicily A. Mirri allows us to define a protocol for stem cells isolation of murine origin. We used 6 Winstar breed male rats whose average weight was 350 g. All animals were sedated with an intramuscular injection of midazolam and anesthesia was maintained with isoflurane and oxygen gas mixture administered with a mask. The adipose tissue has been taken from the root of the animal’s thigh with a small incision. Different steps are needed for processing and digesting the tissue. First it is washed several times in a solution enriched of antibiotics and then is mechanically fragmented. The homogenate is therefore digested enzymatically under permanent shaking. We obtain an heterogeneous population of cells that were subsequently selected through the plastic adhesion. These cells are able to grow and proliferate and show all the characteristics typical of stem cells. In conclusion, we report a multistep and reproducible technique for providing a substantial number of mesenchymal stem cells and for maintaining them in culture.