Human embryonic stem (hES) cells have traditionally been cultured in medium containing fetal calf serum (FCS) and mouse fibroblasts as feeder cells. The use of animal derived materials carries a risk of transmitting animal pathogens, and they are not optimal in cultures aimed at cell transplantation in humans. This technical study aiming at facilitating IVF units to establish new hES cell lines, has systematically compared the non-differentiated growth of the hES cell line HS237, originally derived and thereafter cultured using human foreskin fibroblasts as feeder cells, by culturing it in media containing serum replacement (SR; 10, 15, 20%), FCS, and human serum. In addition, optimal concentrations of insulin-transferrin-selenium (ITS) mixture and the effect of basic fibroblast growth factor (bFGF) have also been studied. Cellular growth was monitored daily and maintenance of their non-differentiated character was studied using antibodies against TRA-1-60, TRA-1-81 and SSEA-4 and expression of Oct-4. The hES cells proliferated fastest when 20% of SR was used. In human serum-containing medium, the cells underwent extensive spontaneous differentiation within a few passages. The FCS supported the non-differentiated growth poorly. Basic fibroblast growth factor supported non-differentiated growth, the highest concentration (8 ng/ml) giving the best result, while ITS was not beneficial.