Stem cell (SC) regenerative therapy represents an emerging strategy for the treatment of human diseases. Since amniotic fluid-derived cells have been recently proposed as a promising source of human SCs, the present research aimed to amplify in vitro and characterize ovine amniotic fluid-derived SCs collected from the membranes (AMSCs) or fluid (AFSCs). These cells were found to proliferate, express the pluripotent SC markers OCT-4 and TERT, and differentiate in both osteogenic and smooth muscle lineages in vitro. However, AMSCs presented an earlier down-regulation of SC markers and a faster rate of differentiation. Thus, AMSCs and AFSCs may represent sources of characterized pluripotent SCs that can be easily collected and amplified in vitro. These ovine SCs may be used in preclinical studies on large animals to develop future human therapies.