Amniotic fluid stem (AFS) cells isolated from human amniotic fluid were cultured and the factors affecting the primary culture of these cells were investigated. The isolated AFS cells express both embryonic stem cell markers, such as Oct-4, and adult stem cell markers, such as CD29, and possess a high proliferation capability. Moreover, isolated AFS cells were able to differentiate into Nestin-positive and α-actin-positive cells. There were several factors, including the date of gestation, the level of blood contamination and the volume of amniotic fluid, which could significantly affect the attachment time and numbers of AFS cells in primary culture. The results showed that the cell attachment time (4.7±0.6 days) during the second trimester of gestation was significantly different from that during the third trimester of gestation (6±0.5 days) (P<0.05), suggesting that cells collected from the fluid during the third trimester of gestation needed a longer attachment time. Blood contamination could significantly affect the cell attachment time. The attachment time of the brown-coloured fluid group (10.8±0.3 days) was significantly different from the blood-cell-free fluid group (6±0.5 days) and the blood-cell group (6.3±0.6 days) (P<0.05). The volume of amniotic fluid influenced, to some extent, both cell attachment numbers and time. With the increase in amniotic fluid volume, cell attachment numbers significantly increased (P<0.05), and cell attachment time extended, but not significantly (P>0.05). The present studies systematically examined the factors that influence the primary culture of human AFS cells and provided useful data on AFS cell research. Additionally, the isolated AFS cells maintain the capacity for differentiation into other cell types and are able to become seed cells for clinical applications.