Human amniotic fluid (AF) contains a variety of stem cells of embryonic and extra-embryonic origins. We characterized two distinct types of stem cells isolated from residual AF material derived from prenatal diagnostic amniocentesis. The two types of cells differed in their morphology and growth kinetics, showing fast (fast human amniotic stem cells; fHASCs) or slow (slow human amniotic stem cells; sHASCs) population-doubling times. Both fHASCs and sHASCs expressed pluripotent stem-cell markers, yet unlike sHASCs, clonogenic fHASCs would generate embryoid bodies and maintain their original phenotype during prolonged in vitro passaging. fHASCs - but not sHASCs - expressed the KLF4, SSEA-4 and CD117 markers. Differential proteomic analysis allowed us to identify the protein patterns specific for either cell type as potentially contributing to their distinct phenotypes. We found thirty-six proteins that were differentially expressed by the two cell types, and those proteins were classified according to their biological and molecular functions. Bioinformatic cluster analysis revealed differential occurrence of cytoskeletal proteins, such as vimentin, F-actin-binding protein, and chloride intracellular channel protein 1. Selected proteins differentially expressed by fHASCs and sHASCs were further characterized by Western blot analysis and confocal microscopy.