Chicken blastodermal cells grown under various culture conditions were characterized by evaluating cell morphology and staining for in vitro markers associated with the ability to contribute to the germline. Staining for alkaline phosphatase (AP) and the embryonic antigen EMA-1 was used to identify cells which had the ability to contribute to the germline, while staining for the embryonic antigen NC-1 was used to identify cells which had differentiated. When chicken blastodermal cells were cultured in medium containing a serum replacement substance (TCM\spTM), more cells stained positive for AP and EMA-1 compared to cultures containing fetal bovine serum or chicken serum. The addition of cytokines known to be beneficial in murine stem and germ cell culture did not increase the number of blastodermal cells staining positive for AP or EMA-1 under the conditions tested, but co-culturing blastodermal cells with Tsai lines 1 or 4, which are derived from 2 day old chick embryos, increased AP and EMA-1 staining and the cells grew in more tightly compacted colonies. The lack of effect of certain cytokines under the conditions tested and the beneficial effect of cell lines derived from 2 day old chicken embryo cells on the growth of EMA-1 and AP positive chicken blastodermal cells indicates that undefined growth factors stimulate proliferation of chicken blastodermal cells. Culturing chicken blastodermal cells on a shaker encouraged the proliferation of a different cell type compared to those produced in other culture systems. The cells proliferating in shaking cultures were AP and EMA-1 positive and grew in cell aggregates rather than attaching to the plate or feeder layer. The cell morphology and staining in the shaking cultures suggests a novel culture method for chicken blastodermal cells which may have the ability to contribute to the germline.