Cellular cardiomyoplasty is undergoing intensive investigation as a new form of therapy for severely damaged hearts. Recent demonstrations confirmed the amniotic fluid (AF) as a putative new source of multipotent stem cells (SC). As a first step before the use of an animal model of myocardial infarction, we investigated whether these human cells could be efficiently differentiate into myogenic lineage in vitro and integrate skeletal muscle in SCID mice. C/kit immunomagnetic-sorted AF cells (AF c/kit+ SC) were characterized by immunocytochemistry and Western blotting for myogenic and early cardiac markers (desmin, MyoD). In vitro, AF c/kit+ SC phenotypic conversion was assayed by myogenic-specific induction media. 1×106 AF c/kit+ SC without ex vivo manipulation were injected in the tibialis anterior muscle of immuno-defficient SCID mice. Acquisition of a myogenic-like phenotype (desmin, MyoD) in AF c/kit+ SC was observed after culture in myogenic-specific induction media. In vivo, transplanted AF c/kit+ SC showed an engraftment in the skeletal muscle of SCID mice but with an unexpected differentiation in tubular gland tissue. No cellular rejection, inflammatory response or tumorogeneicity of these cells was shown. This study showed the ability of these cells to proliferate extensively and transdifferentiate in vitro into the myogenic lineage. Indeed, we showed that these AF c/kit+ SC possess a high myogenic potential with the expression of cytoskeletal markers MyoD and desmin well known to play an important role in cardiac and skeletal muscle function. Nevertheless, despite no immuno-rejection, our results also suggested that ex-vivo engagement in a specific pathway may be needed to induce the differentiation of AF c/kit+ SC into a desired cell lineage before cell transplantation therapy. Overall, our data suggest that AF c/kit+ SC represents a new and very promising source for cell therapy and their potential use in cellular cardiomyoplasty.