Congenital malformations are the leading cause of deaths during the neonatal period. Infants with prenatally diagnosed soft tissue defects may benefit from readily available autologous tissue for surgical implantation perinatally. In this study we investigated the cell content of amniotic fluid (AF) and its suitability for isolation and expansion of autologous cells for clinical use. Second trimester AF was obtained at routine amniocentesis at mean gestational week+day 16+2 (n=54). To investigate the cell content, freshly harvested AF cells (AFC) were analyzed for different cell lineages by flow cytometry. To evaluate the isolation and expansion potential of AFC, isolation by plastic adherence was evaluated with three cell culture media and positive selection of CD117, CD133, CD271, and fibroblast cells were evaluated with minimum essential medium alpha culture media. Both the positive (+) and the negative (-) fractions were analyzed. Surface expression, senescence, immunogenicity, colony forming, proliferation, and differentiation capacity was examined. In the nonhematopoietic, nonendothelial fraction of freshly harvested AF (n=17), 0.13% cells were CD73(+)/CD117(+) and 0.12% CD73(+)/CD271(+). AF displayed large donor variability with varying isolation and expansion success (n=30). The proliferative, differentiative, colony forming capacity and time before senescence also showed high variance. AFC had a preference for osteogenic rather than adipogenic lineages. Most culture-expanded AFC expressed mesenchymal but not hematopoietic surface epitopes. AFC expanded in Dulbecco's modified Eagle's medium showed higher immunogenic characteristics. This study shows that AF is a heterogeneous cell source, with high donor variation. Therefore, it may not be the best source for autologous cell therapy.