Amniotic fluid (AF) contains heterogeneous and multipotential cell types. A pure mesenchymal stem cells group can be sorted from AF using flow cytometry. In order to evaluate a possible therapeutic application of these cells, the human AF-derived c-kit(+) stem cells (c-kit(+) AFS) were compared with the c-kit(-) (unselected) stem cells (c-kit(-) AFS). Our findings revealed that the optimal period to obtain c-kit(+) AFS cells was between 16 and 22 weeks of gestation. Following cell sorting, c-kit(+) AFS cells shared similar morphological and proliferative characteristics as the c-kit(-) AFS cells. Both c-kit(+) and c-kit(-) AFS cells had the characteristics of mesenchymal stem cells through surface marker identification by flow cytometric and immunocytochemical analysis. Both c-kit(+) and c-kit(-) AFS cells could differentiate along adipogenic and osteogenic lineages. However, the myocardial differentiation capacity was enhanced in c-kit(+) AFS cells by detecting GATA-4, cTnT, α-actin, Cx43 mRNA and protein expression after myocardial induction; whereas induced c-kit(-) AFS cells were only detected with GATA-4 mRNA and protein expression. The c-kit(+) AFS cells could have potential clinical application for myogenesis in cardiac regenerative therapy.